FEMS Microbiology Letters 48 (1987) 89-92 89 Published by Elsevier

FEM 02954

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

F r a n k Driehuis and Jan T.M. Woute r s *

Laboratorium voor Microbiologie, Universiteit van Amsterdam, Amsterdam, The Netherlands

Received 7 July 1987 Accepted 10 July 1987

Key words: Escherichia coli; Penicillin-binding protein; (Growth rate)

1. SUMMARY

In an Escherichia coli strain, the levels of peni- cillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth rate in chemostat cultures under different nutrient limitation conditions. A consid- erable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.

2. INTRODUCTION

Seven proteins that bind penicillin covalently are present in the cell envelope of Escherichia coli. These PBPs have been identified as penicillin-sen- sitive enzymes involved in the final stages of peptidoglycan biosynthesis, the polymerisation of the disaccharide pentapeptide precursors into the

Correspondence to: F. Driehuis, Laboratorium voor Microbi- ologie, Universiteit van Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam. The netherlands * Present Address: Nederlands Instituut voor Zuivelon-

derzoek, Postbus 20, 6710 BA Ede, The Netherlands

glycan chains (transglycosylation) and the cross- linking of peptide side-chains (transpeptidation) [1,2]. PBP 1A and PBP 1B are considered to be the major transglycosylase/transpeptidases with a similar and interchangeable role in peptidoglycan synthesis [1,3]. For PBP 3, also a transglyco- sylase/transpeptidase, a specific role in cell divi- sion is assumed [1,4]. PBP 5 and PBP 6 both possess D-alanine carboxypeptidase activity [5], but although these proteins are the most abundant PBP species, they are not essential, at least under laboratory conditions [1,6]. Furthermore, several peptidoglycan hydrolases have been demonstrated in E. coli, assumed to be required for the incorpo- ration of new peptidoglycan in the sacculus [7].

The action of the synthetic and lytic enzymes participating in peptidoglycan biosynthesis has to be coordinated to ensure proper growth, shape and division of the sacculus. In addition, since the peptidoglycan content of E. coli cells is not sig- nificantly affected by the growth rate [8,9], the in vivo rate of peptidoglycan synthesis has to be adjusted to the growth rate. Unfortunately, little information is yet available on the regulatory mechanisms that control peptidoglycan metabo- lism. One approach that might give some insight into this complex matter would be to investigate

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how cellular levels of enzymes involved in pepti- doglycan metabolism vary with growth rate. Re- cently, the effect of growth rate on the cyto- plasmic steps of peptidoglycan synthesis in E. coli was investigated [8]. In this paper we examine the relation between growth rate and the cellular con- tent of PBPs in E. coli. To vary growth rate, chemostat cultures were used. In a previous paper we reported on the effect of growth rate on the composition of peptidoglycan [10].

2. MATERIALS AND METHODS

3.1. Bacterial strain and growth conditions The strain used in this study was E. coli W7

(lysA dapA) [11]. Cells were cultivated in minimal medium in chemostat or batch cultures as de- scribed before [10].

3.2. Detection of PBPs Cells (0.2-0.8 g dry weight) were harvested by

centrifugation at 6000 x g for 10 min and washed once with 10 mM sodium phosphate buffer (pH 7.0). Cell envelopes for the penicillin-binding as- say were prepared as described [12], except that cell breakage was accomplished using a French pressure cell (12000 psi; 4°C) instead of sonica- tion. The cell envelopes were stored in 50 mM sodium phosphate buffer (pH 7.0) at - 20 ° C.

PBPs were detected by incubation of the cell envelope preparations with a saturating concentra- tion of [t4C]benzylpenicillin (2.00 GBq/mmol , 54 mCi/mmol; Amersham), separation of the sarko- syl-soluble proteins by sodium dodecyl sulphate (SDS)-polyacrylamide slab gel electrophoresis on 10% polyacrylamide gels, and finally, visualisation of the radioactively labelled proteins by fluo- rography. This standard procedure for the detec- tion of PBPs is described in detail by Spratt [12]. Equal amounts of protein (approx. 200 /~g) were loaded into the gel slots. X-ray films (Kodak X-Omat XRP-1) were pre-fogged to obtain a lin- ear relationship between the image density on the X-ray film and the amount of radioactivity ap- plied [13]. Quantitation of the PBPs was accom- plished by scanning the fluorographs with a

densitometer (Bio-Rad Model 1600) coupled to an integrator (Spectra-Physics SP4270).

4. RESULTS

Growth conditions such as medium composi- tion and growth phase are known to affect the content of PBPs in E. coli [14,15]. Continuous cultivation in chemostat cultures allows variation of the growth rate while other parameters are kept constant. We used this technique to investigate the relation between the growth rate and the levels of the individual PBPs.

The strain used in this study, E. coli W7, was cultivated in glucose-, phosphate-, or magnesium- limited chemostat cultures at growth rates varying from 0.1 to 0.8 h 1 (equivalent to generation times of 416 and 52 min, respectively). PBPs in cell envelope fractions were examined according standard procedures. Fig. 1 shows an example of the PBP profile obtained in these experiments. PBP 1A and PBP 1B were poorly resolved in our gel system. Since, for unknown reasons, no sep- aration of PBP 5 and PBP 6 was obtained on some gels, we will use the nomenclature PBP 1A/B and PBP 5 /6 in the remainder of this report.

PBP 6 5 3 2 1A/B

t

Fig. 1. PBP profile of E. coli W7. Cell envelopes were in- cubated with [a4C]benzylpenicillin and the labelled proteins were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. The fluorograph and the corresponding densi- tometer tracing show the PBPs from cells grown in batch culture. The tracing of PBP 5 and 6 is obtained from the same piece of gel after a shorter time of exposure to X-ray film.

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Table 1

Relative abundance of PBPs in cell envelopes of E. coli W7 at high growth rate in chemostat or batch cultures

Limiting Growth component rate in medium (h- 1 )

Relative amount of bound [14 C]benzylpenicillin a

PBP 1A/B PBP 2 PBP 3 PBP 4 PBP 5/6

_ b 1.1 63 11 12 17 340 (14.2%) (2.5%) (2.7%) (3.9%) (76.7%)

Glucose 0.8 69 9 9 9 249 (19.9%) (2.7%) (2.7%) (2.7%) (72.0%)

Phosphate 0.8 136 15 19 34 457 (20.2%) (2.2%) (2.9%) (5.2%) (69.4%)

Magnesium 0.8 132 14 17 20 876 (12.5%) (1.3%) (1.6%) (1.9%) (82.7%)

a The amount of bound [14C]benzylpenicillin is expressed in arbitrary units determined by integration of densitometer tracings of fluorographs. Values between brackets give the percentage of bound [a4c]benzylpenicillin for each PBP under the growth condition in question.

b Cells grown in batch culture.

B o t h the g r o w t h rate a n d the k ind o f nutr ient l i m i t a t i o n w e r e f o u n d to i n f l u e n c e PBP levels. T h e

latter aspect is i l lustrated in T a b l e 1 for fas t -grow- ing cells. For c o m p a r i s o n , data f r o m cel ls g r o w n in ba tch cul ture in the s a m e m e d i u m were also

i n c l u d e d . A l l PBPs were present at h igher levels u n d e r p h o s p h a t e - or m a g n e s i u m - l i m i t e d c o n d i - t ions than under g l u c o s e - l i m i t e d or batch cul ture

c o n d i t i o n s . Fig. 2 i l lustrates the e f fect o f the i m - p o s e d g r o w t h rate on the PBP levels . S imi lar ob-

servat ions were m a d e for the 3 nutr ient l imi ta - t ions. T h e a m o u n t o f PBP 1 A / B w a s f o u n d to be i n d e p e n d e n t o f the i m p o s e d g r o w t h rate u n d e r g lucose- or m a g n e s i u m - l i m i t e d c o n d i t i o n s . U n d e r

p h o s p h a t e l imi ta t ion , a reduc t ion o f a b o u t 40% w a s observed at the l o w e s t growth rate. T h e

c D

2oo >,

m

~, 12o

D o

_c 4O

c

~ 24

O

~ g

N

m

P B P 1 A / B 20

4

P B P 4 1500

= 90o A

30C

P B P 2 2c

P B P 5 / 6 500

300

100

P B P 3

S P B P 6

P B P 5

02 O4 O6 O8 O2 O4 O6 08 O.2 04 O6 O8

G r o w t h r a t e ( h - ~ )

Fig. 2. Relative abundance of E. coli W7 PBPs at different growth rates. Cells were grown in glucose-limited (I), phosphate-limited (e), or magnesium-limited (A) chemostat cultures at growth rates varying from 0.1 to 0.8 h-1. PBPs were quantitated as described in the text. Experiments were done in duplicate. Results shown for PBPs 1A/B, 2, 3, 4, and 5/6 are from one typical experiment. The lower right-hand panel shows the results of PBP 5 (e) and PBP 6 (©) separately, under phosphate-limited conditions.

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amounts of PBPs 2, 3 and 4 steadily decreased as the growth rate decreased (with the exception of PBP 4 under glucose limitation). This effect was the most p ronounced in case of PBP 3, for which a 4- to 5-fold reduction was observed. The level of PBP 5 / 6 showed a different pattern. Under the nutrient limitation conditions tested, approx. 25% more PBP 5 / 6 was detected at a growth rate of 0.1 h -1 than at a growth rate of 0.8 h -1. Gels that had allowed separation of PBP 5 and PBP 6 revealed that this increase was due to a 2- to 3-fold increase in PBP 6, while PBP 5 decreased 20-40%. In Fig. 2 this is illustrated for phos- phate-limited cells.

5. D I S C U S S I O N

The data presented here showed that the detec- table amount of the major pept idoglycan transgly- cosylase/ t ranspept idase (PBP 1 A / B ) in E. coli is relatively independent of the growth rate. This observation implies that, at least in cells growing at submaximal rate, these enzymes would be in excess. There also seems to be a surplus of PBPs at maximal growth rate; it has recently been dem- onstrated that E. coli can grow at the max imum growth rate with much less than the normal com- plement of PBPs [16]. F rom these findings it can be concluded that the rate of pept idoglycan synthesis is not regulated by variation in the cellu- lar content of PBPs. This agrees with the results of Wientjes et al. [17], who observed no variation in the cellular content of PBPs during the cell cycle of E. coli, and concluded that the topographical and temporal control of pept idoglycan synthesis is not exerted by regulating the amount of PBPs. Also, the cytoplasmic enzymes catalysing the for- mat ion of uridine nucleotide precursors of pepti- doglycan have been shown to be independent of the growth rate [8].

Slow-growing cells are characterized by a high level of PBP 6 and a low level of PBP 3. The same was previously observed for s tat ionary-phase cells [14]. The high level of PBP 6 in slow-growing or stat ionary-phase cells might possibly provide a clue to the still obscure role of this protein in peptidoglycan metabolism.

Some of our results are in marked contrast with

those reported recently by Tuomanen et al. [18], who also examined PBPs of E. coli W7 at differ- ent growth rates. The amount of detectable PBP 1 A / B decreased steadily as the growth rate de- creased, while the amounts of PBP 3 and PBP 5 / 6 remained relatively constant. However, it should be noted that the PBP profile obtained by these authors exhibited a marked difference f rom the standard PBP profile, in that about 20% of the radioactive penicillin was found to be associated with PBP 4, while a value of about 4% is usually found [14] (Table 1). We do not know the reason for this increased binding to PBP 4, but it may be relevant to the discrepancy between these results and those of Tuomanen et al. [18].

R E F E R E N C E S

[1] Spratt, B.G. (1983) J. Gen. Microbiol. 129, 1247-1260. [2] Matsuhashi, M., Nakagawa, J., Tomioka, S., Ishino, F.

and Tamaki, S. (1982) in Drug Resistance in Bacteria-- Genetics, Biochemistry and Molecular Biology (Mitsu- hashi, S., Ed.) pp. 297-310. Japan Scientific Press, Tokyo.

[3] Yousif, S.Y., Broome-Smith, J.K. and Spratt, B.G. (1985) J. Gen. Microbiol. 131, 2839-2845.

[4] Spratt, B.G. (1975) Proc. Natl. Acad. Sci. USA 72, 2999-3003.

[5] Amanuma, H. and Strominger, J.L. (1980) J. Biol. Chem. 255, 11173-11180.

[6] Broome-Smith, J.K. (1985) J. Gen. Microbiol. 131, 2115-2118.

[7] H~ltje, J.-V. and Schwarz, U. (1985) Molecular Cytology of Escherichia coli (Nanninga, N., Ed.) pp. 77-119. Academic Press, London.

[8] Mengin-Lecreulx, D. and Heijenoort, J. (1985) J. Bacteriol. 163, 208-2122.

[9] Zaritsky, A., Woldringh, C.L. and Mirelman, D. (1979) FEBS Lett. 98, 29-32.

[10] Driehuis, F. and Wouters, J.T.M. (1987) J. Bacteriol. 169, 97-101.

[11] Hartmann, R., H~51tje, J.-V. and Schwarz, U. (1972) Na- ture 235,426-429.

[12] Spratt, B.G. (1977) Eur. J. Biochem. 72, 341-352. [13] Laskey, R.A. and Mills, A.D. (1975) Eur. J. Biochem. 56,

335-341. [14] Buchanan, C.E. and Sowell, M.O. (1982) J. Bacteriol. 151,

491-494. [15] De la Rosa, E.J., de Pedro, M.A. and Vazquez, D. (1982)

FEMS Microbiol. Lett. 14, 91-94. [16] Tuomanen, E. (1986) J. Bacteriol. 167, 535-543. [17] Wientjes, F.B., Olijhoek, T.J.M. Schwarz, U. and Nan-

ninga, N. (1983) J. Bacteriol. 153, 1287-1293. [18] Tuomanen, E., Cozens, R., Tosch, W., Zak, O. and Tomasz,

A. (1986) J. Gen. Microbiol. 132, 1297-1304.