Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae

Vol. 8 1, No. 3, 1978 April 14, 1978

BIOCHEMICAL

AND BIOPHYSICAL

EVIDENCE FOR PEPTIDOGLYCAN-ASSOCIATED IN Neisseria B.H.

RESEARCH COMMUNICATIONS Pages lnll-1017

PROTEIN(S)

gonorrhoeae

S.A. Morse, 192 w. Wong, and F.E.

Hebeler,

Young

Department of Microbiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642 and 1Department of Microbiology and Immunology of Oregon Health Sciences Center, Portland,

University

Received

February

28,

Oregon

97201

1978

Summary: Growth pH markedly influenced the composition of the cell envelope of Neisseria gonorrhoeae. The composition of the peptidoglycan from cells grown at pH 7.2 and 8.0 consisted primarily (91%) of muramic acid, glutamic acid, alanine, meso-diaminopimelic acid, and glucosamine in approximate molar ratios of 1:1:2:1:1. The peptidoglycan from cells grown at pH 6.0 contained an accessory protein(s) which accounted for 42% of the weight of the isolated complex. The chemical grown

confirmed

muramic

cosamine

gonococcal

acid,

glutamic

in approximate

bacteria,

chemotype

thus

I (3,

In cells

grown

identified

Neisseria

gonorrhoeae

acid,

molar

alanine,

ratios

far

examined

by Hebeler

(2).

Both

of 1:1:2:1:1,

(3,

is

groups

acid

of all

4, 5) and probably

(1)

and

of investigators

respectively.

typical

from cells

and Young

meso-diaminopimelic

of the gonococcus

at 37" C in medium buffered in association (1,

peptidoglycans

tine,

and threonine

amino

acids

doglycan

et al.

peptidoglycan

and glu-

In this

re-

the gram-nega-

belongs

to group

5).

mers were

gonococcal

reported

by Wolf-Watz

the peptidoglycan

spect, tive

of purified

at pH 7.2 and 37" C was initially

subsequently found

composition

accounted

preparations.

2To whom correspondence

were for

2).

with

It

should

indicated

that

present less Similar should

(1); than

the peptidoglycan be noted minor

however, 1 percent

results

at pH 7.2,

were

that

amounts

no accessory component

amino

acid

of aspartic

on a weight

basis,

of the dry weight reported

by Wolf-Wats

polyof

analyses acid,

of gly-

these of the peptiet al.

(2).

be addressed.

1011

OOO6-291X/78/0813-lOll$Ol,OO/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 81, No. 3, 1978

In this

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

communication,

composition

we report

of the peptidoglycan

that

the pH of growth

with

respect

can modify

to the presence

the

chemical

of accessory

polymers. MATERIALS AND METHODS Growth conditions and media. Strains of N. gonorrhoeae were grown in liquid gonococcal broth (6) buffered at pH 6.0; 7.2, or 8.0 with N-P-hydroxymethylpiperazine-N'-2-ethanesulfonic acid at a concentration of 30 mM. After final adjustment of the pH with HCl or NaOH, the medium was sterilized by autoclaving and then supplemented with NaHC03 (420 mg/l), glucose (5 g/l) and IsoVitaleX enrichment (BBL) (1% v/v). All cultures were grown and harvested at mid-logarithmic phase as described previously (6). Isolation of peptidoglycan. The peptidoglycan of N. gonorrhoeae was isolated as previously described (7). Essentially, harvested cells were boiled in 4 percent sodium dodecyl sulfate (SDS) for 3 hours and allowed to stand overnight at room temperature. The digest was then centrifuged at 60,000 x g for 45 min and the pellet (peptidoglycan) extensively washed by centrifugation with water. Analytical methods. The amino acid and amino sugar analysis was performed on preparations of purified peptidoglycan hydrolyzed in 4 N HCl at 105' C for 14 hours on a Beckman model 119 amino acid analyzer as described previously (7). Samples of purified peptidoglycan treated with either trypsin (10 pg/ml) or lysozyme (10 pg/ml) were subjected to SDS-polyacrylamide gel electrophoresis (7.5% gels) and stained according to the procedure of Segrest and Jackson (8). RESULTS Table tion

1 shows the percent

as a function

parent

increase

pH 6.0 ranged either

from

mainly

the amount

studies

have demonstrated

far

isolated isolated

that

was not due to an overproduction due to the presence

to dissociate

examined,

from from

cells

cells

SDS-polyacrylamide (within cells

an Rf of 0.1). grown

protein(s).

by additional

The peptidoglycan-protein gel

extractions complex

electrophoresis Suspensions

at pH 6.0 decreased

the apparent

fracthe ap-

grown

grown

increase

of peptidoglycan

of an associated

the protein(s)

unsuccessful.

from

fraction

thus

peptidoglycan

at

at

pH 7.2 or 8.0.

tidoglycan

gel

2 to 8 times

of the apparent

In the strains

pH.

of the peptidoglycan

Subsequent

after

of growth

dry weight

also

per se, Repeated

in boiling remained

and was found

1012

density

following

but was

attempts 4% SDS were

associated

at the

of the peptidoglycan-protein in optical

in pep-

top of the complex treatment

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 8 1, No. 3, 1978

Table Effect

of Growth

1

pH on the Apparent

Content

of Neisseria

gonorrhoeaea

Peptidoglycan Strain

8.0

F62

NDb

1.4

13.4

Jw31

1.9

2.5

11.4

RUG 50

ND

1.1

4.5

cs7

ND

9.2

18.5

10 pg/ml

in optical

of lysozyme,

trypsin,

isolated

from

strain

isolated

amounts

centrations

of all

ratio

when treated

was performed RUG 40 grown cells

grown

of amino

acids

beyond

consisted

isolated

accessory

from

by weight, protein(s)

and

cells with

at pH 8.0,

amino

acids

grown except

of the complex (Table

of the various

grown

Suspen-

at pH 7.2 or 8.0

lysozyme.

7.2,

and 6.0

at pH 8.0 or 7.2 did those

found

at pH 6.0 cystine isolated

from

contained

these

There

was not

amino

acids

of the protein(s).

a consistent

2).

contain

sig-

portion grown

3).

significant (Table cells

of at pH

(Table

and methionine from

(Table

cells

respectively

3).

1013

not

com-

in the peptide

complex

of 9.5% and 9.1% protein

from cells

or pronase.

on the peptidoglycan-protein

The peptidoglycan-protein

The complex

42X,

only

from

peptidoglycan.

in Materials

chymotrypsin,

complex

density analysis

8.0 and pH 7.2

molar

as described

determined.

acid

nificant

About

was isolated

An amino

The complex

@I 6.0

1.0

of the peptidoglycan-protein

plex

the

pH 7.2

0.9

either

decreased

of dry weight)

RUG 40

b ND = not

sions

(percent

pH 8.0

aPeptidoglycan Methods.

with

Peptidoglycan

con2).

consisted

increase Further

of

in the studies

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Amino

Acid

Composition

Complex

2

of the Peptidoglycan-Protein

of Neisseria

gonorrhoeae pH of Growth

Protein-

RUG 40a Media

h

!Lis Amino acids Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

12.7 6.3 10.4 ND ND 16.9 ND ND 7.2 ND 3.7 13.2 2.8 5.2 6.0 ND ND

PeptidoglycanC Muramic acid Glucosamine Alanine Diaminopimelic Glutamic acid

105.0 102.0 165.0 100.0 98.0

acid

1.46 0.64 0.90 ND ND 1.08 ND ND 0.68 ND 0.41 1.49 0.46 0.76 0.77 ND ND 24.46 16.42 11.70 12.90 12.60

pH 7.2 nmol 8.1 2.9 8.3 ND ND 13.7 NTI ND 13.2 ND 10.5 4.9 ND ND 27.7 2.0 ND 92.0 87.0 161.0 100.0 92.0

pH 6.0 Imlol 0.93 0.29

0.72 ND ND 0.78 ND ND 0.13 ND 1.19 0.55 ND ND 3.55 0.27 ND 21.44 14.01 11.43 12.90 11.87

42.1 17.9 22.3 19.0 17.6 31.0 27.1 ND 15.6 ND 22.4 27.7 8.9 14.5 108.4 44.9 122.4

4.84 1.81 1.94 2.45 1.71 1.82 1.92 ND 1.47 ND 2.53 3.13 1.45 2.13 13.87 6.15 10.09

106.4 84.0 166.0 100.0 100.0

24.70 13.52 11.82 12.90 12.90

aTh e growth and isolation of the peptidoglycan-protein complex from N. gonorrhoeae strain RUG 40 was as described in Materials and Methods. Hydrolysis was performed in 4 N HCl, 105' C for 14 hrs. Correction for components degraded by HCl was obtained from a standard curve of samples hydrolyzed for various periods; the degradation for muramic acid and glucosamine was 36 and 26.5% respectively. b Correction

was made to account

for

the gain

of H20 during

hydrolysis.

'Amino acid analysis was determined by standardizing diaminopimelic acid to 100 nmoles. The typical peptidoglycan components used in this determination were muramic acid, glucosamine, alanine, diaminopimelic acid, and glutamic acid in a 1:1:1.65:1:1 ratio respectively. Components found in the peptidoglycan weresubtracted from the protein portion of the peptidoglycan-complex.

1014

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Percent in

Distribution

3

of Protein

and Peptidoglycan

the Peptidoglycan-Protein

Complex

from E. gonorrhoeae

Isolated

RUG 40a

Fractionb Growth pH

Peptidoglycan

(X)

Protein

8.0

90.5

9.5

7.2

90.9

9.1

6.0

57.8

42.2

(X)

aThe growth and isolation of the peptidoglycan-protein complex from N. gonorrhoeae RUG 40 was as described in Materials and Methods. b Percent of each fraction as calculated from data

are required lipid

is

to determine

associated

with

was determined in Table 2.

how many proteins

are

by weight

actually

present

and whether

the protein(s).

DISCUSSION The chemical

composition

on the macromolecular 1965,

changes

acids, were

determined uranic

sicca

in the

for

of growth cell

major

acid medium

of gram-negative

by changes

in the

content phase

growth

(10).

surface.

and some minor

subtilis

of polymers, could

cell

and the carbon

of Bacillus

shifts

teichoic

of the growth

envelope

wall

medium has a significant

of the bacterial

in the galactosamine

that

acid

content the

content

as a function noted

of the growth

(9).

As early variations

More recently,

Similarly,

by limiting it

has been

such as Aerobacter

conditions

1015

(11,

12).

as in amino

in the medium,

such as the substitution

be produced

organisms

source

effect

it

was

of teich-

the phosphorus possible aerogenes

to vary or -N.

Vol. 81,No.3,1978

Although fered

many organisms

medium during

of the effect gonorrhoeae study dant

BIOCHEMICAL

can grow

represents

that

doglycan

isolated

does not

appear

certain

ion

there

to be strain this

grown

one protein

at pH 6.0.

specific.

increment

the consequences

in the protein

and whether

amino

acid

on the atten-

presented

content

it

is

content

the protein(s)

clearly

of the peptithis

effect

not possible reflects

varies

E.

the present

Furthermore,

At present,

in the

While

(13),

The data

buf-

to an analysis

envelope.

5.8 to 8.0

envelope.

increase

from gonococci

whether

in more than

a major

from

to analyze cell

the pH of poorly

on the cell

ranging

attempt

in

has been directed

concentration

on the

is

shifts

attention

at pH values

alterations

establish

major

little

the first

metabolic

produce

growth,

of hydrogen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to as-

an increase

among strains

of

gonococci. The exact

physiologic

Currently,

cidated. attached

we favor

to the peptidoglycan

Escherichia whether

coli there

protein

lipid

serves

serves

The increase

currently

in a fashion

with

rate

in anchoring could

that

this

site

for

the outer

be related

of degradation

this

analogous

have not

as the binding

in protein

under

the hypothesis

associated

a role

or a slower

of the protein(s)

Our analyses

(14).

is

peptidoglycan

thesis

significance

remains protein

to the

permitted

protein

the protein.

to either at pH6.0.

is

covalently

lipoprotein

of

us to establish

or what

membrane

to be elu-

residue

in the

Presumably,

this

to the peptidoglycan.

an increased These

rate

possibilities

of synare

investigation.

ACKNOWLEDGEMENTS This

investigation

and AI-11709

(F.E.Y.)

was supported from

the National

by U.S.P.H.S.

Grants

Institute

of Allergy

AI-13571

(S.A.M.)

and Infectious

Diseases. REFERENCES 1. 2. 3.

Hebeler, B.H. and Young, F.E. (1976), J. Bacterial. 126, 1186-1193. S. and Bloom, G.S. (1975), Infect. Wolf-Wats, H., Elmros, T., Normark, Immun. 11, 1332-1341. Ghuysen, J.-M. (1968), Bacterial. Rev. 32, 425-464.

1016

Vol. 81, No. 3, 1978

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Martin, J.P., Fleck, J., Mock, M. and Ghuysen, J.-M. (1973), Eur. J. Biochem. 2, 301-306. Schleifer, K.H. and Kandler, 0. (1972), Bacterial. Rev. 36, 407-477. Wegener, W.S., Hebeler, B.H. and Morse, S.A. (1977), Infect. Immun. Is, 210-219. Hebeler, B.H. and Young, F.E. (1976), J. Bacterial. 126, 1180-1185. Segrest, J.P. and Jackson, R.L. (1972), in --Methods in Enzymol., (V. Ginsburg, ed.), Academic Press, Inc., New York, pp 54-63. Young, F.E. (1965), Nature 9, 104-105. Ellwood, D.C. and Tempest, D.W. (1969), Biochem. J. 111:1-5. Tempest, D.W. and Ellwood, D.C. (1969), Biotech. BioG. 11, 775-783. McDonald, I.J. and Adams, G.A. (1971), J. Gen. Microbial. 65, 201-207. Morse, S.A., Miller, R.D. and Hebeler, B.H. (1977), in The Gonococcus, (R.B. Roberts, ed.), John Wiley and Sons, Inc., New York, pp 213-253. Braun, V. and Bosch, V. (1972), Eur. J. Biochem. 8, 51-69.

1017