gate control ing the cell wall sysn in staph aureus

8/7/2019 Gate Control Ing the Cell Wall Sysn in Staph Aureus

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Molecular Microbiology (2004) 53(4), 12211231 doi:10.1111/j.1365-2958.2004.04200.x

2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 200453412211231Original ArticleThe gate controlling cell wall synthesis in Staphylococcus aureusH. Komatsuzawa

et al.

Accepted 26 April, 2004. *For correspondence. E-mail [email protected]; Tel. 81 82 257 5637; Fax 81 82 257 5639.

The gate controlling cell wall synthesis inStaphylococcus aureus

Hitoshi Komatsuzawa,

1

* Tamaki Fujiwara,

1

Hiromi Nishi,

1

Sakuo Yamada,

2

Masaru Ohara,

1

Nadine McCallum,

3

Brigitte Berger-Bchi

3

and

Motoyuki Sugai

1

1

Department of Bacteriology, Hiroshima University

Graduate School of Biomedical Sciences, Kasumi 1-2-3,

Minami-ku, Hiroshima city, Hiroshima 734-8553, Japan.

2

Department of Microbiology, Kawasaki Medical School,

Matsushima Kurashiki, Okayama 701-0192, Japan.

3

Department of Medical Microbiology, University of Zrich,

Gloriastr. 32, CH-8028 Zrich, Switzerland.

Summary

Glucosamine-6-P occupies a central position between

cell wall synthesis and glycolysis. In the initial steps

leading to peptidoglycan precursor formation glu-

cosamine-6-P is processed sequentially to UDP-N-

acetylglucosamine, while to enter the glycolysis

pathway, glucosamine-6-P is isomerized by NagB to

fructose-6-P. Although we could not demonstrate

NagB activity, nagBinactivation significantly reduced

growth. Mutational analysis showed that NagA was

involved in glucosamine-6-P formation from N-

acetylglucosamine-6-P, and GlmS in that from fruc-

tose-6-P. Inactivation of glmSprevented growth on

glucose as sole carbon source, which resumed after

complementation with N-acetylglucosamine. Tran-

scription of glmSas well as the amount of GlmS was

reduced in the presence of N-acetylglucosamine.

This and the preferential incorporation of N-

acetylglucosamine over glucose into cell wall material

showed that N-acetylglucosamine was used exclu-

sively for cell wall synthesis, while glucose served

both cell wall synthesis and glycolysis. These obser-

vations suggest furthermore GlmS to be the key and

only enzyme leading from glucose to cell wall synthe-

sis in Staphylococcus aureus, and show that thereexists a tight regulation and hierarchy in sugar utili-

zation. Inactivation of nagA, nagBor glmSaffected

the susceptibility of S. aureusto cell wall synthesis

inhibitors, suggesting an interdependence between

efficiency of cell wall precursor formation and resis-

tance levels.

Introduction

Sugars and/or amino sugars are utilized by bacteria for

the synthesis of cell surface structures, and as exogenous

carbon sources for cellular physiology. Amino sugars

such as glucosamine (GlcN) and N-acetylglucosamine

(GlcNAc) are essential components of peptidoglycan,

lipopolysaccharide in Gram-negative bacteria, and lipote-

ichoic acid in Gram-positive bacteria (Coley et al., 1972;

Plumbridge et al., 1993; Raetz, 1996). Various different

sugars are also utilized in cellular metabolic pathways

such as glycolysis (Plumbridge et al., 1993; Plumbridge

and Vimr, 1999). In addition, some bacteria utilize sugars

for capsule or exopolysaccharide production (Rick andSilver, 1996). Therefore, sugars are not only important

substrates for cell physiology but also for virulence. Trans-

port of sugars into bacterial cells is mediated by phospho-

enolpyruvate phosphotransferase systems (PTS) and

non-PTS, although sugar transport by non-PTS also inter-

acts with various PTS proteins (Postma et al., 1993;

Vadeboncoeur and Pelletier, 1997). Several PTS, includ-

ing some specific for glucose, GlcNAc, N-acetylman-

nosamine (ManNAc) and N-acetylneuraminic acid

(NANA), have been reported in different bacteria (Postma

et al., 1993; Plumbridge and Vimr, 1999) indicating that

PTS are sugar-specific. Using PTS, sugars are taken upby bacteria in a phosphorylated form, then processed and

modified before entering metabolic pathways.

It is known that GlcN-6 phosphate (GlcN-6-P), which is

converted to UDP-GlcNAc by the action of GlmM and then

GlmU, is an initial precursor for cell wall biosynthesis,

while fructose-6-P (Fru-6-P) is an initial precursor for gly-

colysis (Mengin-Lecreulx and Heijenoort, 1994; 1996;

Gehring et al., 1996; Jolly et al., 1997). GlmS (Fru-6-P

amidotransferase) catalyses the conversion of Fru-6P to

GlcN-6-P, while NagB (GlcN-6-P deaminase) catalyses

the reverse reaction converting GlcN-6-P to Fru-6-P

(Plumbridge et al., 1993; Plumbridge, 1996; Plumbridge

and Vimr, 1999). GlcN-6-P can also be derived from

GlcNAc-6-P by NagA (GlcNAc-6P deacetylase), and in

Escherichia coliManNAc and NANA can also be con-

verted to GlcN-6-P via GlcNAc-6P (Plumbridge and Vimr,

1999). Therefore, NagA, NagB and GlmS play key roles

in the distribution of sugars to cell wall synthesis and

glycolysis. As GlmS is the key enzyme responsible for the

synthesis of GlcN-6-P from Fru-6-P, inhibitors of GlmS

activity impede cell wall synthesis and have been reported


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H. Komatsuzawa

et al.

2004 Blackwell Publishing Ltd,

Molecular Microbiology

,

53

, 12211231

as antibacterial agents (Bearne, 1996; Chmara

et al

.,

1998; Bearne and Blouin, 2000). In E. coli, the regulation

of these enzymes is coordinated so that in the presence

of amino sugars GlmS expression is decreased, while

NagB and catabolic enzymes are induced (Plumbridge

et al., 1993; Plumbridge and Vimr, 1999). However, in

Gram-positive bacteria, the regulation mechanism

responsible for GlcN-6-P synthesis has not been investi-

gated, although individual factors have been identified in

several species.

In Staphylococcus aureus, several factors associated

with cell wall synthesis have been identified through the

studies of methicillin resistance in methicillin-resistant

Staphylococcus aureus(MRSA), these include the fem

genes, glmM, fmtA-C, fmhBand others (Berger-Bchi

et al., 1989; Maidhof et al., 1991; Henze et al., 1993;

Gustafson et al., 1994; de Lancastre et al., 1994; Jolly

et al., 1997; Glanzmann et al., 1999; Komatsuzawa et al.,

2000; 2001). However, factors responsible for GlcN-6-P

synthesis have not been well characterized, although cor-

responding genes have been identified in the publishedS. aureusgenome sequences (TIGR: http://www.tigr.org).

GlmS activity has been reported to be increased in a

vancomycin-resistant S. aureusin which cell wall thicken-

ing was observed (Cui et al., 2000), but the precise mech-

anism of vancomycin resistance in this strain has not been

established. It is logical, however, that factors affecting the

distribution of sugars to cell wall synthesis are also likely

to have an effect on antimicrobial resistance.

In this study factors responsible for GlcN-6-P synthesis

in S. aureus, NagA, NagB and GlmS were characterized.

The association of these factors with methicillin and van-

comycin resistance in MRSA was also investigated.

Results

Effects of GlcNAc on growth characteristics ofnagA-,

nagB- andglmS-mutants

To analyse the functions of the enzymes involved in GlcN-

6-P metabolism, GlcNAc was added to chemically defined

medium (CDM) medium and the effect on the growth of

the various mutants and on their gene expression was

determined. The basic CDM used for growth throughout

these experiments contained 50 mM glucose. Glucose

taken up by the PTS appears as Glc-6-P in the cell, which

is metabolized to Fru-6-P. Fru-6-P can subsequently either

enter glycolysis or, after conversion by GlmS to GlcN-6-P

then by GlmM to GlcN-1-P, be used for peptidoglycan

synthesis (Fig. 1). Glucose alone was found to be

sufficient to support both glycolysis and peptidoglycan

synthesis in wild-type S. aureusBB270. The nagA-and

nagB-mutants grew slower than the wild type on medium

containing glucose as sole carbon source, whereby nagA

inactivation reduced the growth rate more markedly than

nagBinactivation (Fig. 2A). In contrast, the glmSmutant

was unable to grow on CDM with glucose as the sole

carbon source, but was able to grow when the medium

was complemented with GlcNAc (Fig. 2B), suggesting

that glmSmutants lacked the ability to form cell wall pre-

cursors from glucose. Adding GlcNAc to CDM had noeffect on the growth rate of the wild-type strain BB270 or

on its nagBmutant, but reduced the growth rate of the

nagA mutant.

Effect of GlcNAc and GlnN on the expression ofNagA,

NagB andGlmS

GlcNAc was shown to complement glmSmutants and

allow them to grow in the presence of glucose. Adding

GlcNAc to the growth medium of a wild-type strain,

although not affecting its growth rate, reduced the pro-

moter activity of glmSby almost half (Fig. 3), thus indicat-ing that GlcNAc is channelled, preferentially over glucose

into peptidoglycan synthesis. In contrast, GlcN, which was

not as efficient in restoring the growth of glmSmutants as

GlcNAc (data not shown), had no effect on glmSexpres-

sion when added to the wild-type strain (Fig. 3). Neither

the nagA nor nagBpromoter activities seemed affected

Fig. 1.

Proposed pathway for GlcN-6-P synthe-sis in

S

.

aureus

. The intermediates and genes

encoding the enzymes involved in the GlcN-6-P synthesis and peptidoglycan synthesis areindicated. Sugars and amino sugars are trans-ported into the cell by phosphotransferase sys-

tems (PTS) in a phosphorylated form. GlcN,glucosamine; GlcNAc, N-acetylglucosamine.
http://www.tigr.org/http://www.tigr.org/


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The gate controlling cell wall synthesis in

Staphylococcus aureus 1223

2004 Blackwell Publishing Ltd,

Molecular Microbiology

,

53

, 12211231

by adding either GlcNAc or GlcN to the growth medium.

Western analysis (Fig. 4) of GlmS confirmed that GlcNAc

reduced the GlmS content of wild-type strain and also that

of the nagBmutant, whereas it did not affect the GlmS

content of the nagA mutant, which does not allow further

GlcNAc-6-P metabolism. Similar transcriptional activities

as observed in BB270 were also seen in the RN4220

background (data not shown).

The effects of various sugars on the glmSpromoter

activity were analysed in more detail by using a glmS-

promoterxylE-reporter gene fusion, which was intro-

duced into the wild type. The repressing effect of GlcNAcaddition on the glmSpromoter activity was concentration-

dependent, increasing with concentrations above 0.5 mM

(Fig. 5). Analysis of the effects of various other sugars

showed that ManNAc and to a slightly lesser extent ManN

Fig. 2.

Effect of GlcNAc on the growth of wild type and isogenic

nagA

,

nagB

and

glmS

mutants. Overnight cultures of the wild type,BB270 (circle),

nagA

mutant (square),

nagB

mutant (triangle) and

glmS

(diamond) were inoculated into fresh chemically definedmedium (CDM), in the absence (A); in the presence (B) of 10 mM

N

-

acetylglucosamine. Growth was monitored by measuring the turbidityat OD

660 nm

.

0.05

0.1

1

5

0 5 10 15 20

Time (h)

B

0.05

0.1

1

5

0 5 10 15 20

OD660nm

OD660nm

A

Fig. 3.

Activity of

glmS

-,

nagA

- and

nagB

promoters in the presenceof different sugars. Strain BB270, containing the corresponding pro-moter

xylE

-reporter gene fusions, was grown in chemically defined

medium (CDM) alone, or CDM supplemented with either 10 mM GlcNor 10 mM GlcNAc as indicated. Promoter activities of the correspond-

ing genes, indicated in the last line, represent the results of threeindependent experiments. The bars represent one standard deviation.Differs significantly (*

P


4/11

1224 H. Komatsuzawaet al.

2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 12211231

repressed glmStranscription, whereas mannose and fruc-

tose had no effect. Although GlcN had no visible impact

on the amount of GlmS by Western blots, a slight

decrease in the glmSactivity was nevertheless percepti-

ble (Fig. 5).

GlmS expression during growth

When grown in CDM, GlmS was present in constant

amounts over the entire growth cycle in the wild type, as

well as in the nagA-and nagB-mutants (Fig. 6). Upon

addition of GlcNAc the GlmS concentration was drasticallyreduced in both the wild type and the nagBmutant,

whereas in the nagA mutant it was not affected, corrobo-

rating the observations made above.

GlcNAc and glucose incorporation in wild type

and mutants

We determined the partitioning of glucose into the total

bacterial cell versus that incorporated into cell wall mate-

rial. Total glucose or GlcNAc uptake was gradually

increased in a time-dependent manner (Fig. 7). After

50 min from the addition of labelled compound, the ratio

of labelled glucose or GlcNAc incorporation into cell wallin total cell mass was almost similar. Two hours after

adding labelled glucose, all label appeared in the total

bacterial cell mass, of which approximately 60% had been

incorporated into the cell wall (Fig. 7). In presence of cold

GlcNAc (10 mM), only half of the glucose was incorpo-

rated within 2 h into the bacterial cell mass, and a slightly

smaller proportion of it appeared in the cell wall fraction

(Figs 7 and 8). GlcNAc in contrast appeared mainly in the

cell wall fraction which comprised 7080% of the added

GlcNAc after 2 h growth (Figs 7 and 8). Interestingly the

percentage of appearance of labelled glucose or GlcNAc

in the total cell mass preceded its accumulation into the

cell wall over time.

The effect of GlcNAc addition on glucose incorporation

and partitioning into the cell wall was determined in the

wild type, nagA-, nagB-and glmS-mutants. In nagA-and

nagB-mutants glucose incorporation was somewhat lower

than in the wild type, reflecting their slower growth com-

pared to the parent strain. Addition of GlcNAc reduced

both total glucose incorporation and the percentage of

glucose diverted into the cell wall of the wild type and

nagBmutant. In the nagA mutant there was also less

glucose incorporated, but interestingly, glucose partition-

ing into the cell wall was not affected. These observations

are a logical consequence of our earlier demonstrationthat GlcNAc, while altering the amounts of GlmS pro-

duced in the wild type and nagBmutant, could not repress

glmSin the nagA mutant. GlmS thus plays an important

Fig. 5. Activity of the glmSpromoter in wild-type strain BB270 in the

presence of different sugars. S. aureuswas grown in various concen-trations of N-acetylglucosamine (GlcNAc), glucosamine (GlcN), N-acetylmannosamine (ManNAc), mannosamine (ManN), mannitol(Man) and fructose (Fru), and the XylE activity of the glmS-promoterxylE-indicator gene fusion was determined. The experiments were

repeated three times. Bars represent one standard deviation. Differssignificantly (*P< 0.05) from none to GlcNAc, GlcN, ManNAc, ManN,Man or Fru.

0

2

4

6

8

10

XylEactivity(mUmg1)

none

GlcNAcGlcN

*

* *

*

ManNManNAc FruMan

50 5 05 .5 0.05 5555 (mM)

Fig. 6. Immunoblot analysis of GlmS during growth. Mutants were grown in chemically defined medium (CDM) in the presence and absence ofglucosamine (GlcN) or N-acetylglucosamine (GlcNAc). Cells were harvested at various growth phases at the OD660 indicated.

0.5 0.8 1.0 1.5 2.0 (O/N) 0.5 0.8 1.0 1.5 2.0 (O/N)

+GlcNAcGlcNAc

wild type

nagA

nagB

OD660nm:


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The gate controlling cell wall synthesis inStaphylococcus aureus 1225


role as a gatekeeper of cell wall synthesis, as shown by

the total lack of cell wall material generated from glucose

in the glmSmutant, which had to be supplemented with

GlcNAc for growth (Fig. 8).

The amount of GlcNAc incorporation in the nagB-and

glmS-mutants was almost identical to that of the parent.

Seventy to eighty per cent of GlcNAc taken up by the cell

was incorporated into the cell wall. In the nagA mutant

GlcNAc taken up was significantly reduced and not incor-

porated into the cell wall as expected.

Similar results were obtained when RN4220 and their

mutants were used for the same experiments (data not

shown).

Minimum inhibitory concentration (MIC) of variousantibiotics innagA-, nagB- andglmS-mutants

Because the genes described in this study are associated

with the early stages of cell wall synthesis, the MICs of

various cell wall synthesis inhibitors were determined for

nagA-, nagB-and glmS-mutants, derived from the MRSA

strain BB270 (Table 1). MICs were recorded after 24 h

and again after 48 h incubations to allow for the slow

growing mutants. In the parent strain, BB270, the MICs

for the various antibiotics were almost the same in both

the presence and absence of GlcNAc, although some

MICs were lower in presence of GlcNAc. In HK9968 (nagA

mutant), MICs for methicillin, oxacillin and bacitracin inCDM without GlcNAc were slightly lower than those of the

parent strain. HK9968 grew very slowly in the presence

of GlcNAc, however, the MICs measured at 48 h were

very similar to those obtained from the media without

GlcNAc. In HK9946 (nagBmutant), the MICs in the

absence of GlcNAc were similar to those of the parent

strain, whereas in the presence of GlcNAc, the MICs for

methicillin, oxacillin and teicoplanin were slightly reduced

even at 48 h incubation. In HK9972 (glmSmutant), MICs

could only be determined in the presence of GlcNAc, the

resulting MICs for methicillin and oxacillin were reduced

compared to those of the parent strain.

Discussion

Sugars, once transported into bacterial cells, can be uti-

lized in diverse cellular processes. The exact mechanism

of sugar distribution has, however, not been thoroughly

investigated, especially in Gram-positive bacteria. In this

report we firstly demonstrated the utilization of glucose

and GlcNAc, major carbohydrate moieties, in both cell wall

synthesis and glycolysis in S. aureus. To further investi-

gate the metabolism of sugars for cell wall biosynthesis,

we focused on the three factors NagA, NagB and GlmS,

associated with the synthesis of GlcN-6-P, a precursor for

cell wall peptidoglycan synthesis (Plumbridge et al., 1993;

Plumbridge and Vimr, 1999). Based on the hypothesis

shown in Fig. 1, we investigated the utilization of glucose

and GlcNAc in nagA-, nagB-and glmS-inactivated

mutants. In the nagA mutant, GlcNAc was not incorpo-

rated into the cell wall, indicating that NagA mediates the

conversion of GlcNAc-6P to GlcN-6-P. Therefore, once

GlcNAc entered the cytoplasm of the nagA mutant, it

Fig. 7. Glucose and N-acetylglucosamine (GlcNAc) incorporation instrain BB270. Radiolabelled glucose or GlcNAc was added to thecells growing in chemically defined medium (CDM). After the time

indicated, radioactivity incorporated into whole bacterial cells (shad-owed bars), or cell wall (white bars) was determined. The resultsrepresent values obtained from three independent experiments. The

bars indicate one standard deviation.

14C-glucose incorporation

GlcNAc ()100

80

60

40

20

0Relativetothem

aximumradioactivity(%)

14C-glucose incorporation

GlcNAc (+)100

80

60

40

20

0Relativetothema

ximumradioactivity(%)

3H-GlcNAc incorporation

GlcNAc (+)

Time (min)

100

80

60

40

20

2010 30 40 50 60 90 120

0Relativetothemaximumradioactivity(%)


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could not be utilized for either glycolysis or cell wall syn-

thesis, leading to GlcNAc-6-P accumulation within the

cells. This is likely to account for the poor growth of the

nagA mutant on GlcNAc, as it is known that accumulationof sugar-6-phosphates inhibits growth (Kadner et al.,

1992). GlcNAc was exclusively incorporated into the cell

wall in the wild type, nagB-and glmS-mutants (Fig. 8).

These results suggested that GlcNAc is primarily used for

the cell wall biosynthesis in S. aureus. These results also

implied that nagBdoes not convert GlcN-6-P to Fru-6-P

in the presence of exogenous GlcNAc.

GlmS expression in the nagA mutant was not inhibited

by the presence of GlcNAc (Figs 3 and 4). The nagA

mutant did not allow GlcNAc-6-P metabolism, suggesting

that glmSpromoter activity and thus the GlmS content of

the cells may be regulated by the GlcN-6-P content of thecells, although this postulated signal is apparently not

triggered by GlcN as efficiently as by GlcNAc. The GlcN

uptake system or GlcN metabolism may be different from

that of GlcNAc. Although the gross uptake of glucose was

reduced in the nagA mutant, the incorporation ratio of

glucose into the cell wall did not decrease correspond-

ingly. This probably resulted in a decreased supply of

glucose for glycolysis. This sugar depletion in the glycol-

ysis pathway may also have contributed to the decreased

growth rate of the nagA mutant in the presence of GlcNAc.

In the glmSmutant, glucose could not be used for cell wallsynthesis, hence the glmSmutant could not grow in CDM

containing glucose as a sole carbon source. GlmS expres-

sion was suppressed by the addition of GlcNAc, which is

preferentially used for cell wall synthesis, indicating that

GlmS is a key factor in the distribution of glucose, via Fru-

6P, to glycolysis and cell wall biosynthesis.

We also investigated the effect of various sugars on

GlmS expression (Fig. 5) and demonstrated that some

sugars (ManNAc and mannosamine) also suppressed the

GlmS expression, while others (fructose and mannitol) did

not. These results indicate that S. aureusis able to dis-

tribute the utilization of various sugars for cell wall synthe-sis and glycolysis. In the nagBmutant there was a

decrease in total glucose uptake in both the presence and

absence of GlcNAc, while GlcNAc uptake was not altered.

These results support the hypothesis proposed for

GlcN-6-P synthesis in Fig. 1. This is the first system pro-

posed, in Gram-positive or Gram-negative bacteria, that

describes the overall utilization of glucose and GlcNAc,

Table 1. Minimum inhibitory concentration (MIC) of various antibiotics of nagA-, nagB- and glmS-mutants.

Strain

GlcNAc

(mM)

Oxacillin Methicillin Teicoplanin Vancomycin Fosfomycin

24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

BB270 010

25632

512512

51264

512512

0.50.03

0.50.25

0.50.25

10.5

3216

6464

HK9968 (nagA) 010

128


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for glycolysis and cell wall synthesis (Plumbridge et al.,

1993; Plumbridge and Vimr, 1999). Our results indicate

that the utilization of glucose and GlcNAc is tightly con-

trolled in S. aureus, and that glmSplays a key role in the

distribution of sugars to the different pathways.

The disturbance of sugar distribution by inactivation of

nagA, nagB, or glmSaffected the antibiotic susceptibilities

of MRSA (Table 1), and to a lesser extent methicillin-

sensitive S. aureus(MSSA) (data not shown), to various

cell wall synthesis inhibitors. Notably, the MRSA, BB270-

derived mutants showed a reduction in MICs to b-lactam

antibiotics, compared to those of the MSSA strain. After

24 h incubation, a significant reduction in the MICs of

various antibiotics was observed, while after 48 h, the

MICs of some antibiotics were increased, suggesting that

growth rate of the mutants was significantly reduced by

the addition of cell wall inhibitors.

The findings in this study provide further knowledge

on S. aureuscell physiology, especially in relation to

sugar metabolism. The impact of nagA-, nagB- and

glmS-inactivation on antibiotic susceptibilities, cell growthand cell survival, suggests that these factors are key

roles for sugar distribution, providing sugars to cell wall

or glycolysis.

Experimental procedures

Bacterial strains and plasmids

E. coliand S. aureuswere grown in LuriaBertani (LB) broth

and Trypticase soy broth (TSB), respectively. When neces-

sary, ampicillin (Ap, 100 mg ml-1), chloramphenicol (Cp,

10 mg ml-1) or tetracycline (Tc, 3 mg ml-1) was added to the

medium. Antibiotics were purchased from Sigma ChemicalCo., St. Louis, MO.

Chemically defined medium (CDM)

CDM for S. aureus(Hassain et al., 1992) consisted of the

following five solutions: Solution 1:20.1 g of Na2HPO4-12H2O;

3 g of KH2PO4; 150 mg of L-aspartic acid, L-gultamic acid, L-

isoleucine, L-leucine, L-proline, L-threonine and L-valine;

100 mg of L-alanine, L-arginine, glycine, L-histidine, L-lysine,

L-methionine, L-phenylalanine, L-serine, L-tryptophan and L-

tyrosine; 50 mg of L-cystine; dissolved in 700 ml of distilled

water and adjusted to pH 7.2. Solution 2: 0.1 mg of biotin,

2 mg of nicotinic acid, 2 mg of D-pantothenic acid, 4 mg of

pyridoxal, 4 mg of pyridoxamine dihydrochloride, 2 mg of

riboflavin, 2 mg of thiamin hydrochloride, dissolved in 100 ml

of distilled water. Solution 3: 20 mg of adenine sulphate and

20 mg of guanine hydrochloride, dissolved in 0.1 M HCl and

made up to 50 ml with distilled water. Solution 4: 10 mg of

CaCl2-6H2O, 5 mg of MnSO4 and 3 mg of (NH4)2SO4-FeSO4-

6H2O, dissolved in 10 ml of 0.1 M HCl. Solution 5: 10 g of

glucose and 500 mg of MgSO4-7H2O, dissolved in 100 ml of

distilled water. Solutions 14 were mixed and the volume

adjusted to 900 ml with distilled water. This mixed solution

and Solution 5 were autoclaved separately, then mixed. When

stated, GlcNAc and GlcN were added to CDM at final con-

centration of 10 mM.

DNA manipulations

Routine DNA manipulations, Southern blotting and hybridiza-

tion were performed according to standard protocols (Sam-

brook et al., 1989). DNA sequencing was performed as

previously described (Komatsuzawa et al., 2000). Poly-

merase chain reaction (PCR) reagents were from Boehringer

Mannheim, and PCR was performed with the GeneAmp PCR

System 2400 (Perkin Elmer). Primers used in this study are

listed in Table 2.

Identification ofnagA, nagB, glmS genes

The DNA sequences of genes encoding putative NagA,NagB, and GlmS of S. aureuswere obtained from the public

database (TIGR: http//http://www.tigr.org) by homology

searches, using the respective amino acid sequences from

E. coli. Based on the DNA sequences, primers were con-

structed to amplify the genes of interest using chromosomal

DNA from S. aureusCOL. PCR fragments were cloned into

pGEM-T easy vector (Promega, Tokyo, Japan) to generate

pHK4435, pHK4445 and pHK4506 (Table 3). The DNA

sequences from both strands of the cloned fragments were

then confirmed.

Construction of recombinant proteins

Recombinant proteins of NagA, NagB and GlmS were

expressed as FLAG-tagged proteins (NagA and GlmS) or as

a His-tagged protein (NagB). Primers used in this study are

listed in Table 2. DNA fragments encoding the protein, ampli-

fied with NagA-5 and NagA-6 for nagA, GlmS-3 and GlmS-4

for glmS, NagB-2 and NagB-3 for nagB, were cloned into

pGEM-T easy vector to generate pHK4454, pHK4456 and

pHK4505, respectively. DNA fragments were then ligated into

the pFLAG MAC vector (Eastman Kodak Company, New

Haven, CT) or pQE30 vector (QIAGEN, Tokyo, Japan) to

generate pHK4466 (nagA), pHK4467 (glmS) and pHK4508

Table 2. Primers used in this study.

sNagA-5 aaa agc tta tgg atg ggt cat acg atsNagA-6 atg aat tca tga tta ttt att agc taNagA-7 gga tcc ttc att acc ttt cat acgNagA-8 cgt gtt aac ggt agc ttaNagA-9 tta agc tta tcg tat gac cca tcc atNagB-1 tta cca cct agt ttg tNagB-2 tgg agc tca ttg gcg tac cga ttt ctNagB-3 tgg gat cca tga aag tat taa act ta

NagB-4 aag ctt aaa cgg cgt acc aggNagB-5 ttg gat cct gta tta aat ttg tgg agNagB-6 gga agc ttc att gtt tac atgFg-1 tta aat aaa gtg tca tcaFg-2 gta aaa ttg ttc cat ccasGlms-3 ata agc tta tgg agg aaa atg tcg ttsGlms-4 tcg aat tca gtg aat tat tcc aca gtGlms-8 gga tcc tag ctt gta cct gct gcGlms-10 ggg gat ccc gac gac caa ttc cGlms-11 gga agc ttc ata ttg gaa tag
http://www.tigr.org/http://www.tigr.org/


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samples were then prepared with the method described

above.

Analysis of promoter activities ofnagA, nagB andglmS

using thexylE-reporter gene

The putative promoter regions of nagA, nagBand glmS were

ligated into the xylEtranscriptional fusion vector pSL24

(Komatsuzawa et al., 1999b). DNA fragments containing the

putative promoter regions were amplified with primers gen-

erating BamHI and HindIII restriction sites at the ends, frag-

ments were then digested and cloned into the pGEM-T easy

vector. The fragments were checked by DNA sequencing

before being excised from the plasmids and ligated into

pSL24 to generate pHK4528 (glmS), pHK4529 (nagB), and

pHK4563 (nagA). The recombinant plasmids were then elec-

troporated into S. aureusRN4220 to generate HK9918

(glmS), HK9920 (nagB) and HK9985 (nagA). Each plasmid

was then transduced into strain BB270 by phage 80alpha to

generate HK10041 (glmS), HK10043 (nagA) and HK10042

(nagB). Strains were grown in CDM supplemented with var-

ious sugars, GlcNAc (0.0550 mM), GlcN (5, 10 mM), Man-

NAc (5 mM), mannosamine (ManN; 5 mM), mannitol (Man;5 mM) and fructose (Fru; 5 mM) as indicated. Cells were

harvested at OD660 nm= 1.0, and whole cell lysates were pre-

pared as previously described (Komatsuzawa et al., 1999b).

The Catechol 2,3-dideoxygenase assay was performed as

previously described (Komatsuzawa et al., 1999b).

Generation ofnagA, nagB andglmS mutants by

Campbell-type integration

Mutants were constructed by Campbell-type integration.

Briefly, DNA fragments containing internal regions of each

open reading frame (ORF) were amplified and cloned into

the pGEM-T easy vector. DNA fragments were then excisedand ligated into the pCL52.1 vector (Komatsuzawa et al.,

1999b), a vector with an S. aureustemperature-sensitive

origin of replication, which can replicate at 30 C but not at

42C. Resulting plasmids were then electroporated into S.

aureusRN4220, and then each plasmid was transduced into

BB270 by phage 80alpha (Komatsuzawa et al., 1999a).

Obtaining bacteria (RN4220 and BB270 derivatives) were

grown at 30C with Tc (10 mg ml-1) to an OD660 nm of 0.2, then

incubated at 42C overnight. A small sample of the culture

at 42C was inoculated into fresh TSB containing Tc

(10 mg ml-1), and incubated at 42C overnight. The appropri-

ate dilutions of the cultures were plated on TS agar plates

containing Tc (10 mg ml-1) and incubated at 42C overnight.

Ten colonies from each electroporation were selected andreplated on TS agar containing Tc. The disruption of the

target genes was confirmed by PCR. In some experiments,

Western blot analysis was also performed to confirm the

absence of functional protein.

Growth rate of the mutants

Overnight cultures of the mutants, grown in CDM, were har-

vested and then washed once with CDM. Aliquots of this

suspension (1 108 cells) were then used to inoculated 10 ml

of CDM broths with or without GlcNAc (10 mM). The growth

was monitored by the measurement of the turbidity at

OD660 nm.

Incorporation of glucose and GlcNAc by mutants

Overnight cultures of the mutants grown in CDM were used

to inoculate fresh CDM with or without GlcNAc (10 mM).Cultures were then incubated at 37C. When the OD660 nmreached 0.4, 3 ml of 14C-glucose (200 mCi ml-1) or 4 ml of 3H-

GlcNAc (1 mCi ml-1) was added. Two samples were then

taken from each of the cultures. One of the samples was

centrifuged; the cell pellet was washed twice with PBS, and

then suspended in PBS. The radioactivity of the cell suspen-

sion was measured to determine the incorporation of the

sugar into the whole cells. The second sample was centri-

fuged and the pellet was resuspended with digestion buffer

[30% raffinose in 50 mM Tris (pH 7.5) with 0.145 M NaCl]

containing 200 mg ml-1 lysostaphin (Sigma Chemical Co., St.

Louis, MO, USA), 10 mg ml-1 of DNase I (Sigma Chemical

Co.) and phenylmethylsulfonyl fluoride (1 mM). The cell sus-

pension was incubated at 37C for 30 min to promote proto-plast formation. The protoplasts were then removed by

centrifugation at 3000 gfor 10 min, and the supernatant was

collected. The radioactivity of the supernatant was measured

to determine sugar incorporation into the cell wall.

MIC testing

MICs of various antibiotics were determined by the microdi-

lution method as described elsewhere (Komatsuzawa et al.,

2000).

Acknowledgements

This work was supported by a grant-in-aid for scientific

research from the Ministry of Education, Science, Sports and

Culture of Japan (14570238). N.M. is supported by the Swiss

National Science Foundation Grant No. NRP4049-06321 to

B.B.-B.

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