THE JOURNAL OF BIOLOGIC.\L CIIE.\IISTRY Vol. 248, No. 6, Issue of March 25, PP. 2161-2169, 1973

Printed in U.S.A.

Turnover of Bacterial Cell Wall Peptidoglycans*

(Received for publication, June 22, 1972)

DEREK BOOTHBY, LOLITA DANEO-MOORE, n!rICHAEL I,. HIGGINS, JACQUES COYETTE, AND GERALD D. SHOCKMAN

From the Departvzellt of Microbiology and Imnzunology, Temple 17~~ice~sify School of Medicine, Philadelphia, Pennsylvania 19l.JO

SUMMARY

The cell wall peptidoglycan of LactobaciZZus acido$hiZus strain 63 AM Gasser has been shown to turn over rapidly during balanced exponential growth and recovery from amino acid deprivation. In contrast, turnover of either pulse- labeled or extensively labeled peptidoglycan of Streptococcus fuecalis ATCC 9790 was not detected by the same method and experimental conditions. In S. faecalis, peptidoglycan turnover could not be induced either by brief exposure of the cells to a low concentration of lysozyme or by growth in the presence of a low concentration of penicillin. The peptido- glycan turnover rate of rapidly growing cultures of L. aci- dophilus was equivalent to a loss of about one third of their peptidoglycan per generation. This was not due to cellular lysis since turnover of protein was below detectable levels. Comparably rapid peptidoglycan turnover rates per generation were observed in slowly growing cultures (30% lost per generation) and in a mutant which autolyzed at about one fifth the rate of the wild type (25% lost per generation). In cultures labeled for 6 or more generations, turnover was preceded by a lag equivalent to 0.8 to 2 generation times. Turnover of pulses of less than 0.2 generations was not ob- served for periods exceeding 2 generations. These last two results cannot be interpreted as indicating that newly made wall is immune to turnover since in both cases nonsynchro- nized cultures were used, and the short pulses would even- tually age as the culture grew. The addition of chloram- phenicol or deprivation of valine completely prevented pep- tidoglycan turnover.

The absence of detectable peptidoglycan turnover in S. faecalis (and other species) suggests that turnover is not an essential feature of cell wall growth. On the other hand, in L. acidophilus, peptidoglycan turnover appears to be closely associated with that portion of wall synthesis related to wall (and surface) enlargement and not to the portion related to wall thickening.

* This investigation was supported by Research Grant AI- 05044 from the National Institute of Allergy and Infectious Dis- eases, United States Public Health Service, and in part by General Research Support Grant 5-50-l-RR-05417 to Temple University School of Medicine from the United States Public Health Service.

Evidence consistent with t.urnover of the cell wall peptido- glycan (mucopeptide, murein) has been obtained with Bacillus megaterium 1U.K (l&3), Bacillus cereus (3), and Bacillus subtilis W23 (2, 4). On the other hand, turnover was not detected during vegetative growth of an 01, E-diaminopimelic acid and I?-sillc-rc,quiring mutant of B. megaterium (5), nor did selective turnover of that peptidoglycan made during tryptophan starvn- tion OCCII~ upon regrowth of B. subtilis try- (6). Pcptidoglycan turnover also has not been observed in Escherichia coli (7, 8).

The absence of pcptidoglycan turnover in some species and its occurrence in others indicates that turnover is not an csscntial feature of peptidoglycan biosynthesis, tither of the type that results in an enlargement in cellular surface area (wall elonga- tion), or that leading to an increase in thickness of the wall (wall thickening) (S). However, in bacterial species that exhibit peptidoglgcan turnover, the phenomenon must bc accounted for in terms of the economy of the entire cell as well as of pepti- doglycan biosynthesis. For esamplc, in some way turnover must be regulated and coupled with other cellular processes so that, in growing and dividing cultures, cellular lysis is not a Significant event. In addit’ion, along with the two known types of wall biosynthesis (thickening and elongation), the turnover process must bc understood in order to interpret data concerning the mode of growth of the cell surface (9). Precise data con- cerning the kinetics of ccl1 wall biosynthesis and degradation have been limited by the absence of suitable methods for the precise, rapid, and convenient determination of the incorporation of radioactive precursors into this cell fraction. Pitel and Gil- varg (5) were able to accomplish this by determining the incor- poration of diaminopimelic acid into cold acid-precipitable ma- terial by using a suitable and well-characterized diaminopimelic acid-requiring mutant. Their method is clearly applicable to such a mutant. With bacterial species and strains which do not contain specific metabolic blocks, diarninopimelic acid may to some extent be converted to lysine and thus find its way into protein. Similar problems may occur with other amino acids that arc components of pcptidoglycan but not of protein (e.g. n-alanine). The importance of this factor to studies of peptido- glycan turnover has been recently pointed out (3). In such cases, as well as in t.hose in which specific peptidoglycan precur- sors arc not utilized by the cell because of permeability problems or, for example, the presence of lysine instead of diaminopimelic acid in the peptidoglycan, a method is required which specifically

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determines the incorporation of a compound into, or release from, the peptidoglycan fraction itself. Such a method does not require exclusive incorporation of the precursor int.0 peptido- glycan and permits parallel studies of its incorporation into other cellular macromolecules (e.g. protein).

Recently, we developed a modification of the Park and Han- cock (10) method for the isolation of a peptidoglycan fraction suitable for handling a large number of samples (11). With bot,h bacterial species studied, X. faecalis ATCC 9790 and L. aci- dophilus strain 63 AM Gasser, this method results in a peptido- glycan fraction that is contaminated with, at most, 7:~; protein. We have now applied this method to a study of peptidoglycan biosynthesis and turnover in these two bacterial species. These species seemed to us to be appropriate for such a study since both contain only one detectable autolytic enzyme of t’he same specificity (a P-A-acetylmuramidase) (12, 13).

EXPERIMENTAL PROCEDURES

Growth of Organisms-S. faecalis ATCC 9790 was grown in a completely defined medium (14), with the following variations: valine-limited medium and threonine-limited medium cont.ained 6 pg per ml of valine and 6 pg per ml of threonine, respectively, instead of the full amounts (100 pg per ml). In order to facili- tate the uptake and incorporation of radioactive lysine, cold lysine in the medium was 30 pg per ml instead of the full amount (110 pg per ml). This quantity is more than sufficient to insure an adequate supply of lysine during the experiments.

A typical label and chase experiment was carried out as fol- lows. Cells were grown for at least eight generations in the presence of both L-['~C]- and [3H]lysine (0.2 PCi of Q4C]lysine, 1.0 PC1 of L-[3H]lysine, and 30 pg of unlabeled lysine per ml). At the desired time, the cultures were chilled, harvested by centrifugation, and resuspended in growth medium containing [3H]- but not [‘%]lysine (10 PC1 of 3H and 300 pg of unlabeled lysine per ml). In this way, the specific activity of that 3H label still present was maintained constant (e.g. 0.033 /Xi of 3H per pg of lysine). In some experiments, the labeling and chase were carried out in the same tube by the addition of a lo- or, more frequently, at least a loo-fold excess of unlabeled lysine.

L. acidophilus strain 63 AM Gasser was also grown in a com- pletely synthetic medium modified from that of Soska (15, 16). For valine starvation experiments, the organism was grown to a turbidity equivalent to 0.24 mg per ml dry weight in complete medium, collected by filtration, washed, and resuspended in medium from which valine was omitted. Specific act’ivities and amino acid concentrations were the same as in experiments with S. faecalis.

Peptidoglycan Determination-Samples (0.5 ml) were removed at intervals into 2 ml of 10% chilled trichloroacetic acid. When all of the samples had been taken, the tubes were heated at 96’ for 30 min in a waterbath. The contents of the tubes were col- lected on glass fiber filters (Reeve Angel No. 984H, 2.4.cm diame- ter) and washed successively with 10% trichloroacetic acid (5 x 5 ml), 10% trichloroacetic acid containing 1 mg per ml of cold L-lysine (2 ml), 75% ethanol (2 ml), and Tris buffer, pH 7.8 (2 ml). The filters were then transferred to 5-ml disposable plastic beakers and incubated with Pronase (2 mg in 2 ml of 0.05 M Tris buffer, pH 7.8) for 30 min at 37”. Following incu- bation, the filters were washed with 4 ml of distilled water and then by 4 ml of absolute ethanol, air-dried, and transferred to scintillation vials. NCS (0.5 ml, Amersham-Searle) was added to each vial and incubated at room temperature for 30 min; 5 ml of scintillation fluid (4 g of 2,5-diphenyloxazole and 0.1 g of

1,4-bis-2-(5.phenyloxazolyl)-benzene per liter of toluene) was added, and the samples were counted in a Nuclear-Chicago Mark I scintillation counter set for dual labels. Efficiency was about 50% for 14C and 20% for 3H, with about 15yc of l?C in the 3H channel and 0.2% SE1 in the 14C channel. Counts were corrected and converted to disintegrations per min (dpm) per 0.5 ml sample.

Incorporation of Labels into Total Acid-precipitable illaterial- Samples collected as described above were washed 5 times with 5 ml of cold 10% trichloroacetic acid and counted. Results obtained with X. faecalis with a leucine label give the same rate of incorporation as those with a lysine label. Thus, in the ab- sence of peptidoglycan turnover, the incorporation of lysine into total acid-precipitable material can be used as an index of protein synthesis, although it is really measuring protein plus peptidoglycan synthesis. In the cases where incorporation of lysine into peptidoglycan influenced the slope of the curve, the corresponding peptidoglycan values have been subtracted to give true protein.

Electron LVicroscopq-Cells were fixed for 2 hours at room temperature in 3% glutaraldehyde, postfixed in osmium tetros- ide, infiltrated and embedded in Epon 812, and poststained in uranyl acetate and lead citrate as described previously (17).

RESULTS

Experiments with Streptococcus faecalis

Exponential Phase (Log) Cells-Exponential-phase cells were inoculated into complete medium containing both [‘“Cl- and [3H]lysine (0.2 $Zi of [14C]lysine, 1.0 PCi of [3H]lysine, and 30 pg of unlabeled lysine per ml). The culture was allowed to grow to a turbidity equivalent to 60 pg per ml dry weight and was then used to inoculate a tube of identical medium at a low turbidity (dilution 1 to 100). This was to allow at least six doublings to occur in order to insure complete equilibration of both labels. At a turbidity equivalent to 40 pg per ml dry weight, the tube was chilled, and the cells were collected by cen- trifugation, suspended in 1 ml of chilled medium, and inoculated into complete medium containing [3H]- but not [14C]lysinc (10 &I of 3H- and 300 pg of unlabeled lysine per ml). At frequent intervals, samples were removed for the determination of radio- activity in both the total acid-precipitable fraction and the pep- tidoglycan fraction. As shown in Fig. l-4, [3H]lysine was in- corporated into both total acid-precipitable material and the peptidoglycan fraction at the same rate as the increase in mass of the culture (as measured by turbidity). The [14C]lysine label, which was absent from time zero in Fig. 19, increased somewhat in the peptidoglycan fraction. This increase was possibly due to a less than adequate chase of the old label neces- sitated by the double label technique, although a similar rise in 14C in total acid-precipitable material was not observed. These results indicate the absence of turnover of either protein (tot,al) or peptidoglycan in extensively labeled log cells of S. faecalis over a period equivalent to about seven generations. Similar results were obtained when the labels were reversed or when [14C]leucine was used as a label for protein and [3H]lysine for peptidoglycan.

Recovery from Amino Acid Starvation-Walls of cells starved for an essential amino acid are thicker than those of exponentially growing cells (17). Furthermore, electron microscopic observa- tions suggested that a small amount of wall hydrolysis may have occurred during regrowth of S. faecalis after 10 hours of amino acid (threonine) starvation (18). It thus seemed possible that

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FIG. 1 (left and center). Peptidoglycan and protein biosyn- thesis and turnover in Streptococcus faecalis during exponential growth (A) and during recovery from 3 hours of valine starvation (B). The increase in turbidity (A), incorporation of a contin- uously present [sH]lysine label into cold trichloroacetic acid- precipitable material (total, 0) and into the peptidoglycan frac- tion (o), and the turnover of a previously present [‘%]lysine label into cold trichloroacetic acid-nrecioitable material ftotal (old), l ) and into the peptidoglycan fraction (R) were deter- mined. Each unit of the turbidity scale on the right is equivalent to about 4 Mg per ml dry weight of cell substance. The dpm scale

turnover may occur during regrowth from these conditions. Valine-limited medium containing I%- and 3H-labeled lysine was inoculated so that at least six generations occurred before starvation commenced. After 3 hours of starvation, the cells were chilled, collected by centrifugation, resuspended in 1 ml of chilled medium, and inoculated into complete medium containing 13H]- but not [r4C]lysine at the same specific activity as the initial growth.

Fig. 1B shows that again the rate of incorporation of the con tinuously present 3H label into total acid-precipitable material and into the peptidoglycan fraction closely paralleled the increase in turbidity of the culture and that no decrease in 14C label in either the total trichloroacetic acid-precipitable material or the peptidoglycan fraction was observed. In both experiments shown in Fig. 1, [3H]lysine in the peptidoglycan fraction was 21 to 247” of the total. This is the percentage expected from preri- ous chemical determinations of the total and cell wall lysine (19). Similar results were obtained with cells recovering from 3 to 10 hours of threonine starvation. Experimental details were the same as those described above.

Turnover of Pulse-labeled Wall-In the experiments described above, cells were labeled for at least six generations before the chase, and, consequently, turnover restricted to newly made wall might not have been detected. In order to investigate the possibility of turnover restricted to a small portion of the wall, cells were pulsed with [3H]lysine for different fractions of a generation and then chased. Cells were grown for about six generations as described above in complete medium containing [14C]lysine of the same specific activity as in previous experiments to insure thorough labeling of the wall. When the culture reached a turbidity equivalent to 40 to 120 pg per ml cellular dry weight, 3-ml samples were added to tubes containing 50 &i of [3H]lysine with no added carrier lysine. At the end of

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on the left is adjusted for the various measurements as indicated on the figure. For example, in A, dpm per 0.5 ml sample of the “total” samples should be multiplied by 102, as indicated on the figure, as well as bv 103. as indicated on the ordinate scale. In - I order to continue the chase for many generations, it was necessary to dilute the cells with fresh medium after 120 min. An adjust- ment for this dilution was made in nlottina the data.

FIG. 2 (right). Peptidoglycan a&l pro&in biosynthesis and turnover in Streptococcus faecalis during recovery from peptido- glycan damage inflicted by lysozyme. Symbols as in Fig. 1.

pulses, of Sia, 36, and >i of a generation time, the cultures were rapidly chilled, collected by centrifugation, resuspended into 1 ml of chilled medium, and inoculated into complete medium contain- ing 110 Kg of unlabeled lysine per ml (no [‘“Cl- or [3H]lysine). In all cases, the cell population during the pulse was comparable, and the experiment was arranged so that pulses of various time intervals terminated at the same time.

In no case was a decrease in the pulsed 3H (or continuous 1%) lysine label in the peptidoglycan fraction observed over a chase period exceeding two generations. The same results were ob- tained in separate experiments using pulses of 1, 5, and 8 min. In some cases a slight rise in the ratio of 3H to 14C was noted during the first 7 to 10 min of chasing. This could be due to a relatively slow conversion of lysine into acid-insoluble peptido- glycan.

Repair of Damaged Wall-It seemed possible that turnover could occur as a function of some sort of a repair mechanism. If this is so, turnover might be demonstrated during the recovery of growth of cells with damaged walls.

Preliminary experiments indicated that cultures could recover from brief treatments with a relatively low concentration of Iysozyme. Cultures continuously labeled with both [‘“Cl- and [3H]1ysine for at least 10 generations as described above were exposed to 0.03 pg per ml of hen egg white Iysozyme (EC 3.2- 1.17) in 0.1 M sodium phosphate buffer (pH 6.7), containing 0.5 M sucrose for 6 min at 37”. A sample (0.5 ml) was then inoculated into the complete growth medium containing t3H]- lysine at the same specific activity as in the previous growth medium. During recovery of growth the turbidity was followed, and samples (0.5 ml) were removed for the determination of radioactivity in the trichloroacetic acid-precipitable and peptido- glycan fractions. As shown in Fig. 2, the initial decrease in turbidity (about 16%) was accompanied by similar drops in

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FIG. 3 (left and center). Peptidoglycan and protein biosyn- thesis and turnover in Lactobacillus acidophilus during ex- ponential growth (A) and during recovery from 5 hours of valine starvation (B). Symbols as in Fig. 1. The dpm scale on the left is adjusted for the various measurements as indicated in the legend for Fig. 1. In the case of the old label, values for the total incorporation of [‘4C]lysine were converted to protein by sub- tracting the corresponding values for the peptidoglycan fraction.

FIG. 4 (right). Comparison of the lag before turnover and

both continuously present (3H) and old (1°C) label in both the total trichloroacetic acid-precipitable and peptidoglycan frac- tions. This initial loss of label can be accounted for by Iysis of a small portion of the cell population. The initial loss was fol- lowed by increases in the continuously present (W) label which paralleled the increase in turbidity, and by no loss in the old (‘“C) label. The small rise in W, especially in the peptidoglycan fraction, could be due to the incorporation of low molecular weight precursors from the lysed cells.

We have also failed to detect turnover in cells recovering from 3 hours of threonine starvation in the presence of penicillin (0.5 pg per ml added 35 min after the start of recovery). TJnder these conditions, turbidity, prot,ein, and peptidoglycan all increase normally but the cells tend to be rod-shaped and dis- tortcd.1

Experiments with Lactobacillus acidophilus

Synthesis and Turnover during Exponential Growth and Re- covery jrom starvation-Cultures of L. acidophilus were grown and labeled as indicated above for S. faecalis. As shown in Fig. 3, incorporation of continuously present [3H]lysine into protein and peptidoglycan closely followed the increase in tur- bidity of both the exponentially growing culture (Fig. 38) and that recovering from 5 hours of valine starvation (Fig. 3B). Also, in both cases turnover of protein, previously labeled with [14C]1ysine, was not observed. Turnover of peptidoglycan, after a lag of about 1 hour (approximately equivalent to one doubling time for mass, protein or peptidoglycan) in the log culture and about 1 to 135 hours in the cultures recovering from valine starvation was observed. After its onset, turnover oc- curred with half-lives of about 110 and 175 min and for about two and one generations for the log and recovering cultures, respectively. The rate of turnover of the log culture is equiva- lent to a loss of about one third of its peptidoglycan per genera-

1 S. Scott and G. D. Shockman, unpublished observations.

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the rate of t,urnover of peptidoglycan synthesized during expo- nent,ial growt,h (LOG) wit,h that made during 3 hours of valine deprivation (STATIONARY), when Lactobacillus acidophilus is permitted to regrow. A, increase in turbidity of the cult,ure re- covering from 3 hours of valine starvation. B, fate of the l4C- labeled peptidoglycan that had been made during the previous exponential growth phase (0) and the 3H-labeled peptidoglycan that had been made during the 3.hollr period of valine starvation (0). For details, see the text.

t.ion. In both cases turnorer slowed and then stopped at. a high cell densitv when the increase in mass of the cultures slowed and stopped. ‘It is also of interest to note that the esponcntial rate of incorporation of the continuously present [%]lysinc label was the same during the lag period before turnover and when turnover of pcptidoglycan was observed (Fig. 3A).

The relationship of turnover to cellular growth was also demon- strated by t’hc addition of chloramphenicol (60 pg per ml) to the chase medium. Cells were lab&d for over eight generations with [Yllysine as described above and chased into 10 volumes of unlabeled medium, with and without chloramphenicol. In the case of chlorarnphenicol, turnover of the previously lnbcled peptidoglycan was rcduccd to zero from a half-life of 130 min (lag before turnover 50 min). I’cptidoglycan biosynthesis continued after chlorztrnpl~c~~icol addit.ion at a To of about 110 min (compared to a Tu of 67 min in the control) until the amount of pcptidoglycan nearly doubled at about 100 min. After t’hat time pcpt’idoglycnn biosynthesis slowed and quickly crnsed. Also, this concentration of chloramphenicol was accompanied by an immediate decrease in the rate of turbidity increase. IIOW- ever, over the course of the experiment (200 min) the turbidit. of t.ho treated culture rose slowly by about 60yc, whercns t’he untreated culture increased about IO-fold. Turnover of protein was not observed in either culture. Similarly, peptidoglycan turnover was completely prevented by valine deprivation and t,he addition of chloralrll)hrnicol 1 hour after the start of the chase.

Xiowly -4utoZyzing Xulant-Mauck et al. (2) suggest that the autolytic N-acetylmulalll?-I-L-alalliilc amidase of both B. sub- tilis W23 and B. megaterium KM play important roles in pepti- doglycnn turnover. To examine the relationship of autolytic activity to peptidoglycan turnover, a mutant of L. acidophilus which nutolyzes at less than 207, of the rate of the wild type in 0.01 M sodium citrate buffer, isolated by Dr. R. Joseph in this laboratory, was examined for peptidoglycan turnover. The rates of growth and protein and pcptidoglycan synthesis were

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little different from the wild type (Table I). After a lag of provides a gap of one generation betmccn the old l*C label and similar length to that observed with the wild type, peptidoglycan the pulsed 3H label. In both types of experiments (Table II), turnover, at a rate only slightly slower than observed in wild evidence for a lag of at least 0.8 of a generation time before turn- type cultures, was observed. Turnover of protein was not seen. over commenced was observed. For the pulsed 3H label, longer

Slowly Growing Cells-In the complete defined medium L. lags were seen. In fact, with pulses of 10 min (about 0.2 of a acidophilus grows with a doubling time of 54 to 75 min. When L-alaninc and pyridoxamine arc omitted from the growth me- dium or when the medium contains a reduced concentration of valine (5 pg per ml), exponential growth is slowed to a 140- t,o 155-min doubling time. As summarized in Table I, in these

TABLE II

Turnover of pulse and extensively labeled peptidoglycan in L. acidophilus

-

I 1 - I Expel.

merit no.”

Pulse time i- Doubling

time (2’0) (mass)

Half-life of peptidoglycan

Lag before t”lTlO”e*

- Relatiw

to TD

r clss pe :ener- ation

II Min it

Wative o TD of

mass

slowly growing cultures peptidoglycan turnover still occurred. The rates of protein and peptidoglycan synthesis mere again very similar to the growth rate. Although peptidoglycan turn- over occurred at slower absolute rates, the percentages of peptido- &can lost per generation (29 and 31yo per generation) wcrc ~ within the range of those seen for the more rapidly growing log cultures (average 33 A 5% per generation). 1

Turnover of “Old” Versus “New” I’eptidoglycan-Mauck et al.

(2) suggest that new wall of B. subtilis W23 is unavailable for knover for about one half of a generation, but that no fraction of the wall is immune from turnover. Also, iii the experiments tipscribed thus far, a lag before turnover equivalent to 0.8 to well over one generation time was observed (Table I).

2 This ~vas un-

espected since extensively labeled cells were employed so that both “old” and “new” wall contained label. It was thought that this could be an artifact of t’hc centrifugation technique employed to ensure a complete and thorough chase. Therefore, - two different methods of chasing old label wcrc utilized. The 3

first used a chase with a IO-fold excess of unlabeled lysinc in the sanle tube, and the second used transfer of centrifuged cells to a second tube containing 3 pg per ml of unlabeled lysine, groITth

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1.5 1.4 1.4 1.6

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- for one generation, followed by Ipulses of [3H]lysinc (1.6 PC1 per 1111) for various timr intervals which were then chased \I-ith at

a Experiments 1 and 2 utilized the chase technique that pro-

lrast a 100-fold excess of 112C]lysine (300 pg per ml). The first vides a one-generation time gap for the old (‘“C) label (see text for details). This has been accounted for in the lag times for the

technique gives the kast disturbance of the cultures, but pro- continuous label. Experiment 3 utilized chases with IO-fold vides only marginal chasing conditions. The second tc&nique excesses of unlabeled lysine in the same tube.

TABLE I

I%osynthesis and turnovrr of extensively labelecl peplidoglycan and protein in L. acidophilus

In all experiments, the cellular mass at the start, of the chase was 20 to 40 pg per ml of cellular dry weight. - Doubling time (To) Lag before turnover

Half-life of Per cent peptidoglycsn lost leptidoglycan per generation

?lZi?z

125 110 120 79 75

120 70

120 130 95 63 83

31 32 33 38 38 26 43 26 25 33 44 26

33 f 5 average

295 290

175

29 31

25

Experiment No.

1 65 2 61 3 71 4 54 5 52 6 52 7 57 8 53 9 52

10 55 11 52 12 53

13 14

15

144 152

73

Cell culture

Log cells

Min 1 1 ielative to PD of mass

58 0.9 50 0.8 60 0.9 80 1.5 65 1.3 70 1.4 60 1.1 55 1.0 77 1.5 95 1.7 60 1.1 75 1.4

115 300

80

0.8 2.0

1.1

milt

75 75 75

70 64 74

163 185

75 78

Slou-ly growing cells (log cells)

Slowly autolyzing mutant (log cells

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generation) or less, no turnover was observed over a period of 335 generations. This does not appear to be an artifact of rapid incorporation of lysine into protein which remains with the pep- tidoglycan fraction since the method was found to remove leu- tine from the peptidoglycan fraction of [14C]leucine pulsed or continuously labeled cells with equal efficiency.

Turnover of Wall Made during Amino Acid Starvation (Wall Thickening) Compared with Turnover of TVall Made during Ex- ponential Growth-Log cells were grown in the presence of [14C]lysine for rx~ly generations as described above. This culture was centrifuged and resuspended in a growth medium from which valine was omitted, in the presence of [3H]lysine. After 3 hours of valine starvation, the cells were again collected, resuspended, and incubated in complctc medium containing no label. As shown in Fig. 4, a similar lag before turnover of bot’h labels (120 and 140 min for the 14C log phase and 3H stationary phase labels, respectively) and comparable rates of turnover (half-lives of 195 and 205 min for the log and stationary phase labels, respectively) were observed.

Effect of Fresh and “Partially Spent” Growth Media-In the experiments described above, chases were performed either by centrifuging the cells and placing them in a fresh growth medium, or by the addition of a high concentration of unlabeled lysine or fresh media containing unlabeled lysine, to the same undisturbed culture tube or flask. With these different chase methods, significant variations in either lag time or turnover rates were not observed. However, partially spent unlabeled media were used for chases in the studies demonstrating peptidoglycan turnover in B. subtilis and B. megaterium (2). Since partially spent media would contain excreted metabolic products, pos- sibly including wall hydrolytic enzymes, this factor was examined. Chases of extensively labeled cells into fresh media or into media in which cells had been grown to a turbidity equivalent to 25 pg per ml cellular dry weight (and resterilized by filtration) resulted in lag times before turnover (70 min) and turnover rates (half- time 90 min) that were indistinguishable from each other.

Ultrastructure of Cell Walls of S. faecalis and L. acidophilus-

In thin sections, the wall of’ L. acidophilus (Fig. 5) does not ap- pear as dense and compact as that of many other gram-positive species (20). Although the walls of S. faecalis characteristically have a dark-light-dark tribanded profile when seen in antitan- gential sections, such is not the case for L. acidophilus walls. Only the very inner portion of the wall, lying adjacent to the plasma membrane, stained densely, whereas the remainder of the wall appeared to be loosely organized and somewhat diffuse (Fig. 5). The wall of log-phase cells (Fig. 5A) appeared to be considerably thinner than that of a cell in the stationary phase after 5 hours of valine deprivation (Fig. 5B). In the absence of a tribanded wall it is extremely difficult, if not impossible, to determine the angle of the section through the wall and therefore to quantitate accurately and to compare average wall thickness. In approximate terms, wall thickness expanded from about 18 to 20 nm in log cells and to 40 to 70 nm in cells deprived of valine for 5 hours. In addition to thinness, walls in log cells (Fig. 5, A and D) showed an extremely diffuse and irregular outer profile, with identifiable pieces of wall very loosely attached and, in some instances, not visibly attached to the bulk of the wall. Under t,hese growth conditions, the labeling experiments de- scribed above (e.g. Fig. 3A) demonstrated peptidoglycan turn- over equivalent to a loss of about one third of the cellular peptido- glycan per mass doubling. In contrast, the walls of cells deprived of valine for 5 hours showed a more easily defined and regular external surface (Fig. 5B). Peptidoglycan turnover was

not observed during the stationary phase due to valine depriva- tion or chloramphenicol treatment. That the apparent slough- ing off of pieces of wall is correlated with peptidoglycan turnover is in contrast to the very regular exterior profile (17, 24) and absence of peptidoglycan turnover seen with S. faecalis under all growth conditions used.

An interesting aspect of the wall thickening process in L. acidophilus is the apparent irregular nature of the thickened wall. Although the outer diameter of the cells was nearly the same along the entire length of cylindrically shaped cells, the diameter of protoplasts, within the wall, fluctuated in a wavelike pat.tern, suggesting a lack of uniformity in the distribution of wall synthetic sites on the cell surface. Similar types of ir- regularity in wall thickening have recent,ly been observed in B. subtilis and B. megaterium by Frehel et al. (21) and in B. cereus by Chung (22). After valine starvation, frequently the poles of the cells and the pads of wall between cells in chains were seen to be extensively thickened.

When thick-walled, valine-deprived cells were permitted to resume growth in a fresh growth medium, several changes in ult,rastructure were seen, typified by the section of a cell shown in Fig. 5C and the higher magnification of a portion of a cell section shown in Fig. 5E, both taken from a culture that had experienced an 0.5 increase in mass. The relatively smooth outer contour of the thickened wall was lost, and a less regular outer contour was seen. Relatively large pieces of wall were seen coming off from the remaining thickened cylindrical portion of the wall, as well as from the thinner (perhaps newer) sections of wall. The sloughing off of pieces of wall makes it impossible to localize the areas of the surface that are engaged in surface enlargement, if indeed these are localized in this species. During such conditions of regrowth, peptidoglycan turnover was ob- served (Fig. 3B). Poles of the cells tended to remain relatively smooth, and many poles retained thickened portions well into the growth recovery process, in agreement with the observations of several species of bacilli (21, 22). Also, the thinning out of cylindrical portions of the wall and elongation of new, thinner cylindrical wall resulted in an apparent expansion of the diameter of thr protoplasm so that the wavelike pattern of wall projecting into the cytoplasm was no longer seen under these areas of the wall. After recovery of growth for 1.5 mass doublings, most cells had thin walls all along the cylindrical portion of the cell, but the poles on some cells were seen to remain thickened.

DISCUSSION

By using the same technique, wall peptidoglycan turnover could not he detected during growth of X. faecalis, whereas it was found to occur in growing, and presumably dividing, cul- tures of L. acidophilus. In both species exponential phase cells are prone to autolysis, whereas stationary phase cells resist autolysis (12, 13). In S. faecalis wall (and peptidoglycan) biosynthesis has been found to occur at a very large number of sites over the ent,ire surface of the coccus (23). Only a limited number of these wall synthetic sites, localized at and near nascent cross walls, could be associated with the process of surface en- largement (24) and with activity of the active form of the auto- lytic enzyme (25). The other peripheral sites appear to be primarily engaged in wall thickening both during normal expo- nential growth (24) and after inhibition of protein synthesis (17). The labeling of peptidoglycan for a small fraction of a generation time followed by a chase for two or more generations should be capable of detecting turnover of a small fraction of

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FIG. 5. Electron micrographs of thin sections of Lactobacillus acidophilus. A, section of a cell from a sample taken from an exponentially growing culture. The wall is rather thin, densely staining only at the very inner portion, and the remainder appears loosely organized with a very irregular outer profile. B, section of a cell taken from a culture that had been deprived of valine for 5 hours. The walls on such cells are thickened, have a fairly smooth and regular outer profile and an irregular wavelike inner profile, suggestive of a lack of uniformity of the wall thickening process on the cell surface. C, section of a cell taken from a cul- ture that had been starved of valine for 5 hours and then allowed

to resume growth in a complete medium until the cell mass had increased by 507,. The wall on this cell remains thickened only at a restricted area of t’he cylindrical portion of the wall and at the pole on t.he right. Note that, like the wall on the starved cell, the diameter of the cell is about the same over its entire length, and the membrane underlying the thickened section of wall ap- pears to be pushed into the cytoplasm. The smoother outer con- tour of the thickened wall appears t,o be lost, except at the cell poles, and pieces of wall appear to be sloughing off from the wall surface. D, higher magnification of a section from an exponen- tial phase culture. The thin, irregular outer contour and ap-

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FIG. 5. D, and E. parent sloughing off of wall pieces can be seen. E, higher mag- nification of a section from a culture recovering from 5 hours of valine deprivation for 0.5 mass doublings. Most of the cylindrical portion of the wall has been thinned out. Still thickened wall

can be seen at the extreme Ze.ft over the nascent cross wall and somewhat thickened and smoother contoured wall on the pole at the right. The bars in A through D equal 100 nm. The bar in D also applies to E.

the total peptidoglycan, either when it is newly synthesized or as it ages in the cell cycle. Turnover was not even detectable during the transition of thick walled, stationary phase cocci to thinner walled, exponential phase cells, where a notching, splitting, and peeling apart of the wall at the base of the now growing cross-wall was seen, along with an over 4-fold increase in the ability of cells to autolyae (18). I f hydrolysis of bonds in the peptidoglycan is important to, or plays a role in, surface enlargement, the hydrolytic process seems to be under exquisite control in S. faecalis, not only during exponential growth, but also during (a) the growth reinitiation process, (b) growth after hen egg white lysozyme damage to the peptidoglycan, and (c) growth in the presence of a concentration of penicillin that fails to affect the rate of growth but does affect the shape of the cells.

The absence of peptidoglycan turnover during growth of at least three bacterial species, S. faecalis, a mutant of B. mega- terium (5), and E. coli (7, 8), suggests that peptidoglycan turn- over is not an essential feature of peptidoglycan biosynthesis per se or wall growth (surface enlargement) in either coccal or rod-shaped bacterial species. On the other hand, the existence of turnover during growth of other species, as well as some of the peculiarities of the process, must be explained in greater detail than simply as differences in species, growth conditions, or techniques used.

In L. acidophilus, peptidoglycan turnover appears to be a very real phenomenon which can occur under a wide variety of growth conditions and in the absence of detectable turnover of protein or lysis of even a relatively small fraction of the cell population. Thus all, or at least most, of the cells in the culture remain intact but lose, on the average, one third of their peptidoglycan per generation (Table I). Turnover occurred after inoculating centrifuged and washed log cells into fresh medium or into par- tially spent medium, upon recovery from amino acid starvation, and upon dilution of label in the same culture tube by the addi- tion of fresh medium containing unlabeled lysine or by adding excess lysine only. Thus, “conditioning” of the medium or cells does not seem to be an important factor. Peptidoglycan turnover occurred equally well in the presence of a high (300

pg per ml) or low (30 pg per ml and less as the available lysine was consumed by the culture) concentration of lysine.

Consistent with results obtained with various species of bacilli (2,3), turnover of peptidoglycan in L. acidophilus seems to occur only when the cultures are growing. In fact, over a a-fold range, the rate of turnover was approximately proportional to growth rate (Table I). However, turnover does not appear to depend on peptidoglycan synthesis per se or, for that matter, on new cell wall deposition (2). Turnover ceased, but peptidoglycan synthesis primarily directed towards wall thickening continued upon valine deprivation or chloramphenicol addition. This sug- gests that turnover is related to the portion of peptidoglycan biosynthesis that results in surface enlargement rather than the portion which results in wall thickening. However, both pep- tidoglycan made during exponential growth and during valine starvation were equally susccptiblc to turnover (Fig. 4).

Although the products of peptidoglycan turnover were not examined in this study, it seems likely that the only detectable peptidoglycan hydrolase, a P-N-acetylmuramidase (13), plays a role. However, peptidoglycan turnover was observed during exponential growth of a mutant that autolyzed at less than 20 % of the rate of the wild type (Table I). This mutant grew only slightly slower than the wild type. At present little is known concerning the regulation of the autolytic enzyme in this species. It is extremely difficult, if not impossible, to quantitate the actual levels of total or functional autolytic enzyme in this species, as it seems to be in most others. In part this is due to its lack of stability, unusual substrate binding properties, and lack of knowledge as to how these enzymes are exported through the membrane (9, 13, 16). It might well be that, irrespective of its enzymatic specificity, only a relatively small portion of the cellu- lar autolytic activity is involved with peptidoglycan turnover during growth and that this portion is unaffected in this mutant.

The ultrastructural observations are consistent with (a) the occurrence of peptidoglycan turnover during growth, and pre- sumably division; (b) the absence of turnover in stationary phase and chloramphenicol-treated cultures in L. acidophilus; and (c)

the absence of detectable peptidoglycan turnover in S. faecalis.

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The appearmcc of t,he walls of growing cells of L. acidophilus appears to bc similar to that seen on growing cells of other lacto- bacilli such as another strain of L. acidophilus (26), L. ptantarum (27)) and L. cccsei (28, 29). In fact, the irregular exterior profile with the apparent sloughing of pieces of wall is also similar to that seen on the wall of s~ernl bacillus species (6, 21, 22, 30), some of which have been shown to turn over their pcptidoglycan (l-4).

The nature of the wall thickening process in L. acidophilus appears to bc less regular than that previously seen in X. jaecalis (17) This wavelike thickening could be the result of a concen- tration of wall synthetic sites at various areas on the surface. This in turn suggests that surface enlargement in this rod-shaped species may not be entirely randorn or diffuse, as has been pro- posed for other grampositive, rod-shaped species (21, 31). On the other hand, it seems that surface enlargement may not be limited to one to three sites per cell as it appears to be in S. jaecalis. A layering of thickened wall was not observed in L.

acidophilus, in contrast to that seen in B. subtilis (6) and B. cereus (22)

Two aspects of peptidoglycan turnover in L. acidophilus re- main to be satisfactorily explained. First is the consistently observed lags of nearly one generation time, observed even upon carrying out the chase in relatively undisturbed and nonsyn- chronized cultures which had been grown in the presence of label for at least sis generations. It cannot be argued that peptido- glycan made at a particular stage of the cell division cycle is immune to turnover, since the growth and labeling conditions would result in extensive labeling of peptidoglycan made during all stages of the past several cell cycles. Part, but not all, of the lag period could be due to an unusually long time required for added unlabeled lysine to equilibrate with intracellular labeled lysine destined to be incorporat.ed into peptidoglycan. Such a delay would result in the continued incorporation of intracellular low molecular weight (labeled) lysine containing compounds (such as the UDP-N-acetylmuramylpeptides) into acid-insoluble peptidoglycan after the chase. In the experiments presented, during the lag period, an increase in radioactivity of the pep- tidoglycan could easily have been masked, or balanced off, by turnover. For example, calculation of some of the data pre- sented would suggest that peptidoglycan turnover commencing at the start of the chase could be balanced by the continued in- corporation of intracellular label for a period of 0.1 to 0.2 gcn- erations, in an exponentially growing culture. This phenomenon or intracellular turnover, or both, could account for the long lags. Preliminary evidence that either or both of these factors were at least in part responsible for the observed lag was obtained in a few experiments which included measurements of the chase of pulse (3H) and continuously present (W) lysine from total tri- chloroacetic acid-precipitable material as well as frorn pepti- doglycan. The data suggest slower incorporation of lysine into peptidoglycan than into protein. Similarly, in order to explain an aspect of their results on the mode of in vivo assembly of the cell wall of B. subtilis, Mauck and Glascr (32) postulated the possibility “. . that certain pools of [cell wall] intermediates are not exhausted during 0.25 generations in unlabeled medium.”

A second unexplained observation, the absence of turnover of pulses of less than one fifth of a generation, may be related to the first. Even if the rate of peptidoglycan synthesis is not uni- form during the cell cycle (33)) it seems unlikely that newly made peptidoglycan could remain immune to turnover as it “ages” during growth and division during a chase of 335 generation

times. A combination of continued incorporation of precursors into peptidoglycan could be balanced by turnover during the early portion of the chase. Alternately, the results could be accounted for by the following hypothesis. Lysine could be incorporated into two peptidoglycan products. One of these products turns over very slowly, or not at all, and is more rapidly labeled. The second product turns over rapidly and becomes labeled more slowly. In this way, during the short pulses most of the [3H]lysine would be incorporated into the product which turns over slowly, whereas longer pulses would result in more of the product which turns over more rapidly.

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4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15. 18.

17.

18.

19.

20.

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22. 23.

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32. 33.

CHALOUPKA, J. (1967) Folia Microbial. 12, 264 MAUCK, J., CH~ZS, L., AND GLASER, L. (1971) J. Biol. Chem.

246, 1820 CHALOUPIOY, J., ANn KROCI~OVA, P. (1971) Folk Microbial.

16, 372 MAUCK, J., ANV GI~.UEII, L. (1970) Biochem. Biophys. Res.

Commun. 39, 699 PITEL,~~. W., .\ND GILV.4RG,C.(l970)~.l~ioZ.Chem. 246, G711 HUGHES, l<.. C., TANXEI~, P. J., AND STOKES, 13. (1970) Bio-

them. J. 120, 159 VAN TUISNRGEN, IX. P., AND SETLOW, It. B. (1961) Biophys. J.

1, 589 ROTHFIELD, L., AND PI,:.\L~A~~-KoTHI~:NC.~, M. (1969) J. Mol.

Biol. 44, 477 HIGGINS, M. L., AND SHOCKMAS, G. 11. (1971) ClZC 0%. IZeu.

Microbial. 1, 29 PARK, J. T., AND Ha~cocrc, R. (1960) J. Gen. Micyobiol. 22,

249 BOOTHUY, ]).,DAN~.;o-MooRE,L., *ND SHOCRMAN,(;.~~. (1971)

Anal. Biochem. 44, 645 SHOCKMAN, G. II., TnoMPso~, J. S., AND Coluovm, M. J.

(1967) Diochemistry 6, 1054 COYETTI:, J., AND Grrurs~.:x, J.-ll/I. (1970) Biochemistr!l 9,

2952 SHOCKMAN, G. L>. (1962) in Analytical MicrobioZog?q (KAVA-

NAGH, F., ed) p. 567, Academic Press, New York SO&A, J. (1966) J. Bacterial. 91, 1840 COYETTX, J., .\ND SHocImm, G. D. (1973) J. Nacteriol.

114, in press HIGGINS, M. L., AXD QHOCKXAT, G. I>. (1970) J. Nacteriol.

103, 244 HIGGINS, M. L., POOLI:Y,I-I. M., AND SHOCKMAN,G. 11. (1971)

J. Bacterial. 106, 1175 TOISNNIES, G., Bamy, B., AND SAOCKMAN, G. D. (1959) J.

BioZ. Chem. 234, 32GB GLAUERT, A. M., AND THORNLEY, R/I. J. (1969) Anwu. Rev.

Microbial. 23, 1525 FRISHEL, C. A., BNAUFILS. A.-M., AND RATER. A. (1971) Ann.

Inst. kasteul: 121, 139 CHUNG. K. L. (1071) Can. J. Microbial. 17. 1565 POOL&, H. R/I:, AND SHOCKMAN, G. D. (1970) J. Hacteriol.

103, 457 HIGGINS, M. I,., AND SHOCKMAN, G. L). (1970) J. fjacteriol.

101, 643 HIGGINS, M. L., PooLEY,H.M., ANI) SHOCKMAN,(:.~). (1970)

J. Bacterial. 103, 504 CHAI, N.-C., AND IARK, K. G. (1970) J. Bacterial. 101, 1005 KAIO;FUDA, T., HOLDISN, J. T., AXD UTECH, N. M. (1967) J.

Bacterial. 93, 472 BEOWN, J. P., EDWARDS,M. IL., ASV VBN DEM~IC, P. J. (1968)

Can. J. Microbial. 14, 823 THORNE, K. J. I., AND BAIZIOZR, D. C. (1969) Eur. J. Biochem.

11, 582 COLE, Ii.. M., POPIiIS, T. J., BOyLAS, It. J., AND lMI,:NDELSOS,

N. H. (1970) J. Uacleriol. 103. 793 MRUCK, j., &AN, L., GLASEE, i., AND WILLI.\MSON, J. (1972)

J. Bacterial. 109, 373 Mt\ucr<, J., ;ZND GLXXR, L. (1972) J. HioZ. Chem. 247, 1180 IIOFFMAN, B., MESSER, W., AND SCII~V~RZ, U. (1972) J. Su-

pramol. Slruc. 1, 29

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D. ShockmanDerek Boothby, Lolita Daneo-Moore, Michael L. Higgins, Jacques Coyette and Gerald

Turnover of Bacterial Cell Wall Peptidoglycans

1973, 248:2161-2169.J. Biol. Chem. 

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