The orthonitrophenyl-beta-~galactosidehydrolase from Pseudomonas aeruginosa (0:11 serotypes) is a superficial enzyme
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
PHILLIPPE POINDRON, ALAINBOURGUIGNAT, FRANGOISE SCHEID, A N D YVESLOMBARD Unit~c~rsi~P Loriis PcrslcJrrr, U . E . R . clc,s Sciences Plicrr~irrrcerr~iqrres, Lcrboraroirc,~de Virologic el Micr.oDiologie gPrlPrnle, 3 , r?re de I'Argorine, F-67083 Srrtrsborrrg, C ~ d e . rFrcrnce ,
Accepted February 17, 1977 POINDRON, P., A. BOURGUIGNAT, F. SCHEID. and Y. LOMBARD. 1977. The orthonitrophenylbeta-D-galactoside hydrolase from Pserrdomortcrs nerrrginoscr (0: I 1 serotypes) is a superficial enzyme. Can. J . Microbiol. 23: 798-810. About 95% of the 0 : l l strains of Pserrdornonrrs trerrrgir~oscr was able to hydrolyze orthonitrophenyl-beta-D-galactopyranoside(ONPG), but was unable to use lactose. The ONPG-hydrolyzing enzyme was located essentially in the periplasm, as seen by biochemicaland ultrastructural studies. POINDRON, P., A. BOURGUIGNAT, F. S C H E I D et Y. LOMBARD. 1977. The orthonitrophenylbeta-D-galactoside hydrolase from Psrrrdornoricrs crerrrginosa (0: 11 serotypes) is a superficial enzyme. Can. J. Microbiol. 23: 798-810. Environ 95% des souches 0:ll de Pserrdomonrcs aerr~ginoscr sont capables d'hydrolyser I'onhonitrophenyl-beta-~-galactopyranoside(ONPG), mais ne peuvent utiliserle lactose comme source de carbone. Pour mieux comprendre le r6le de I'enzyme hydrolysant I'ONPG, les auteurs etudient la localisation de I'enzyme dans la bacterie. Celui-ci est situe essentiellement dans le periplasme, comme le montrent les etudes biochimiques et ultrastructurales.
Introduction The microbiologists admit generally that Pseudorno~iasaeruginosa is unable to hydrolyze orthonitrophenyl - beta - D - galactopyranoside (ONPG). Nevertheless a few years ago, Bulow (4) found that some strains of the species were endowed with this property. We were unaware of this observation when we stated during an epidemiological study (22) that about 95% of the 0 : 11 strains (according to Habs (14) and Veron (28)) synthesized such an enzyme. Since our first study which dealt with 240 strains of all serotypes, we extended this observation to a population of 647 strains originating from several hospitals. Since the 0 : 1 1 strains were unable to grow in lninimal medium added with lactose, the questions of the localization and roles of the ONPGhydrolyzing ~noleculearose. We report here a preliminary response to these questions. Materials and Methods Bac~erialSlrains, Media, and Grobvrh Condirions
The D 72/28 strain of our collection was chosen as representative of 0 : I I serotypes, because it possessed the typical characters of the species and rapidly hydrolyzed ONPG (according to Le Minor and Ben Hamida (17)). The strain was grown either in trypticase soja broth
(TSB) (BD MCrieux) or in brain heart infusion (BHI) (Institut Pasteur Production). The choice of the latter medium was circumstantial; we used i t because the results obtained in TSB depended on the batch used. The CaZ+ and Mg2+ concentrations of the media might be of importance to interpret the results; we measured them by atomic absorption spectrophotometry (TSB batch 1, reference not recorded, divalent cations not determined; TSB batch 2, reference No. 251 1, C a Z +: 28 mg/P, Mg2+: 17.6 mg/P; BHI, reference No. 1318, C a Z +: 16.4 mg/P, Mgzf : 7.6 mg/e). In some cases, we used minimal medium (glucose: 0.0139 M , KZHPOJ: 0.4 M, KHzPOA: 0.022 M , MgS04 and CaCI, adjusted at the required concentration). The bacteria were grown at 37"C, either in a 250-ml flask (100ml of medium under mild agitation (90 to and fro movements per min), or in tubes (14 x 140 mm) (5 ml of medium) under vigorous agitation (1 60 to-and-fro movements per minute). When experiments were carried out in minimal medium, sterilizable polypropylene tubes were used. Asscry of EIIZJJI~IPS
ONPG-hydrolase (ONPGH) was assayed at 30°C according to Dean and Smith (11) except for the incubation period which was longer (I8 h). We shall justify this choice in another paper. One unit (U) corresponds to the amount of enzyme that is able to hydrolyze 1 nmol o n ONPG per minute. Glucose-6-phosphate dehydrogenase (G6PDH, E C 1.1.1.49) is a cytoplasmic enzyme. It was assayed according t o D e Moss (12) to check the leakage of cytoplasmic material in the fractions.
POlNDRON ET AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
Assay of Proteit~s It was performed according to Lowry et 01. (19). Fractionation of tile Bactet.ia1 Cells Unless otherwise mentioned, all operations were carried at 4'C. In most instances, we treated the bacteria according to the method of Gilleland et a/. (13): 10 ml of appropriate culture was centrifuged at 6SOOg for 20 min. The supernatant was discarded, and the pellet was washed in deionized water, and centrifuged again in the same conditions. The pellet was then resuspended in 0.03 M tris(hydroxymethyl)amino methane (Tris) HCI, pH 8.6, containing I niM ethylenediaminetetraacetic acid (EDTA), and 0.6 M sucrose (N-tris(hydroxymethy1)methyl-2-amino-ethanesulfonic acid (TES) 8.6) (80 ml of TES 8.61250 mg of bacteria in wet weight). In someexperiments we used 0.1 m M EDTA instead of 1 m M (TES 8.6; 0.1). The suspension, called 'complete system,' was gently shaken on a rotative shaker for 1 h at room temperature. Its optical density at 660 nm was measured, and referred to as 100%. The suspension was then centrifuged at 6500g for 20 min. The opalescent supernatant (S,6.5) was centrifuged again at 30 0 0 0 g for 10 min. Proteins and enzymes were assayed in the resulting supernatant (S130) and pellet (P,30). TES 8.6 - treated P. aerugitloso are osmotically fragile. They are called for this reason 'osmoplasts' (13). We checked the osmotic fragility of the osmoplast pellet (P,6.5) as follows: P16.5 was rapidly suspended in deionized water and vigorously shaken with a Vortex shaker for exactly 1 min. The optical density at 660 nm of this suspension was measured immediately, and again after 1 h at room temperature. The osmotic fragility was expressed as a percentage of decrease in optical density, by comparison with the optical density of the complete system set at 100%. The shocked suspension was finally centrifuged at 30 0 0 0 g for 20 min. The supernatant (S230) and the pellet (P,30) were assayed for proteins and enzyme. Because of the effect of TES 8.6 treatment on the cytoplasmic membrane of P. aerr~git~osa, we used also a milder procedure to fractionate the bacteria (Neu and Chou (20)). They were treated exactly as above with the following two modifications. (I) The initial washing of the bacteria was made with 0.03 M TrisNaCI, p H 7.3, instead of deionized water. (11) TES 8.6 was replaced by 0.03 M Tris.HCI, pH 7.3, containing 1 m M EDTA and 0.6 M sucrose. Electron-microscopic Observations Ultratl~inSections The samples were fixed for 2 h, at 4'C, with a mixture formed of one volume of 25% (v/v) glutaraldehyde and four volumes of 0.1 M phosphate buffer, pH 7.4. After thorough washings with 0.1 M phosphate buffer, the samples were treated with a mixture formed of one volume of 2 x (w/v) osmic acid and one volume of 0.2 M phosphate buffer. They were preembedded in buffered agarose, dehydrated, and embedded in Epon Araldite. Sections were made with an O M U 2 Reichert ultramicrotome. They were stained with a mixture o f uranyl acetate and lead citrate. Negative stainings These were made, following standard techniques, with potassium phosphotungstate.
799
Results Preliminary Observatiot~son ONPG H Preliminary experiments showed that the shock fluid S,30 from TSB 1 - grown bacteria, treated according to Gilleland er al. (13), had a good ONPGH activity. We therefore used S,30 to check if ONPG-hydrolyzing property is due to an enzyme. Heating at 100°C for 1 h, or treatment for I h at 37°C with 0.25% buffered trypsin, pH 7.3, destroyed the hydrolytic activity. The ONPG hydrolysis by the 0: l l strains can therefore be ascribed to a thermolabile protein. In whole bacteria, enzyme activity was suppressed by toluene or chloroform; in siru lipids might play a role in the normal functionning of ONPGH. We observed also that the enzyme was not stable at 4°C.
I17j7ueiice of the Groit~tl7Medium on the Bekavio~ir of the Bacterial Cells upon T E S 8.6 Treatment Nossal and Heppel (21) have shown that the selective release of enzymes by osmotic shock takes place under milder conditions with exponentially growing cells than with Eschericl?ia coli in stationary phase. We therefore studied first 15-h-old bacteria, assuming that P. aeruginosa which is very sensitive to the combined action of Tris and EDTA would be less fragile in these conditions; we observed that the results depended both on growth conditions and culture medium. Culture in Flask under Mild Agitation (Table 1 ) Table I shows that protein distribution within the various fractions does not depend upon the nutritional conditions imposed to the bacterial cells. This is not the case for ONPGH activity. The S,30 and P,30 fractions from BHI-grown bacteria contained lower levels of ONPGH activity than those from TSB-grown bacteria. With TSB 1, the S,30 contained 160% of the total ONPGH activity of the whole bacteria taken as control. At first sight this result is in favor of a periplasmic localization for ONPGH. With TSB 2, results were heterogeneous: enzyme activity varied from one experiment t o another. It was always low in the S230 from BHI-grown cells. With all media, little, if any, activity was detectable in the P230. In all series of experiments we found cases where no enzyme activity was detectable in the fractions. Since the protein distribution did not
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
800
CAN.
I. MICROBIOL. VOL. 23. 1977
depend on the nutritional conditions, this apparent lack of activity on O N P G H must be related to qualitative (limiting cofactors) rather than quantitatives factors (amount of proteins). In deionized water, BHI osmoplasts behave unexpectedly. The optical density at 660 nm of the complete system did not decrease as with TSB 1 and TSB 2 osmoplasts, but increased. Since G 6 P D H activity was absent in S130 and in S230 from BHI-grown bacteria, we may conclude that the O N P G H activity detected in these fractions originated from superficial sites of the bacterial cell. The comparison between the sum of the O N P G H activity of the four fractions (CAF) and the activity of the control sample (AC) provided further information. The ratio EAF/ A C was about 2.5 in TSB 1 experiments (7 expts.), 1.2 in TSB 2 experiments (4 expts.), and 0.5 in BHI experiments (3 expts.). The enzyme seems to be 'cryptic' in TSB experiments, and loses its activity in the case of BHI-grown cells. Since in both media, a sonicate of washed bacteria had exactly the same specific activity as washed unbroken control bacteria, we may conclude that the apparent crypticity is not due to inaccessibility of the enzyme in vivo, but to another factor which depends chiefly on the culture medium and the fractionation.
this latter enzyme was present in the corresponding fractions from TSB-grown bacteria.
Influence of Age (Table 2) The results observed with 15-h-old bacteria might reflect the physiological state of bacteria sampled a t the beginning of the stationary phase and, more particularly, the heterogeneity of the bacterial population. We studied thus 8-h-old bacteria, grown in tubes under vigorous agitation. In these conditions, the cells had a higher O N P G H specific activity. T h e protein distribution in the fractions was not affected. However, the age influenced the O N P G H distribution. Now the S,30 and the S230 had two t o seven times higher levels of O N P G H activity. With TES 8.6 - treated cells, the mean ratio EAFIAC was about 1 ; with TES 8.6; 0.1 treated-cells, it was about 1.6.
Stin7ulation or Inhibition of the ONPGH Activity The correlation between the serotype and the presence of O N P G H activity, the superficial localization of this enzyme, its sensitivity t o organic solvents, the heterogeneous results observed in certain conditions, the apparent crypticity, o r the apparent loss of the enzyme activity suggested to us (I) that this enzyme might play a role in lipopolysaccharide (LPS) Culture in Tubes under Vigorous Agitntion biosynthesis and (11) that it required several (Tc1ble 2 ) factors to function normally, probably a thermoThe results were analogous but not exactly stable carbohydrate moiety, non-covalentlyidentical with the previous data. We found the linked lipids, and the O N P G H itself. W e have following differences. (I) When grown in TSB, assumed that each of these factors was critical the whole cells had a three-to-four times higher for the hydrolysis reaction. T E S 8.6 treatment O N P G H specific activity whereas this specific would dissociate more or less the components, activity remained unchanged with BHI-grown whose respective concentrations in the fractions bacteria. (11) The protein distribution was dif- might vary, depending on the age and the culture ferent and reflected a greater sensitivity of the conditions. If this hypothesis is correct, recell to TES 8.6 treatment. (Much more proteins combination experiments between crude fracin S,30 and S,30, greater decrease of the tions on the one hand, and heterologous o r optical density of the TSB osmoplasts in de- homologous fractions treated in various ways o n ionized water, smaller increase with BHI osmo- the other one, must reveal modifications in the plasts.) The replacement of TES 8.6 by TES 8.6; enzyme activity of the crude samples used as 0.1 did not change the results except for the source of enzyme. lower sensitivity of the osmoplasts to the hypoIt was possible to stimulate (Table 3) the tonic shock. (111) The ratios CAF/AC were O N P G H activity in S,30 upon addition of a n always of about 0.5. The O N P G H activity is lost equal volume of a homologous preparation, during the fractionations. heated to 60°C, and slowly cooled to 30°C (such The BHI osmoplasts resisted the hypotonic a treatment increases acceptor activity o f shock; there was no G 6 P D H either in the S130 Snlmonelln LPS by allowing its optimal associao r in the S,30 from BHI-grown cells, whereas tion with phospholipids (24, 25)). The heated
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
TABLE 1. Protein, ONPGH, and G6PDH distribution in the fractions obtained from cells grown under mild agitation in balloon flask, treated then according to Gilleland ef al. (13) Control (washed bacteria) Medium TSB batch 1 (15 h) (7 expts.)
Protein" ONPGH" G6PDH"sb
% of control
100
ND
8.90 (k2.8)
-
0.62d (k0.22)
ND
-
100
100
ND
8 (k3.5)
-
0.47 (k0.07)
ND
-
% of control
100
100
100
U/mg
-
0.67 (f0.06)
0.199 (k0.06)
11.2 (k2.3) -
% of control
U/mg
BHI (15 h) (4 expts.)
Protein
100
U/mg TSB batch 2 (15 h) (4 expts.)
S130
nMean values. *Determined o n a sonicate o f bacteria. Cdenotes a decrease. an increase m the O D 660 nm. dstandard deviation. NOTE:N D , not done.
+
ONPGH 2 expts. : 0 5 expts.: 51 (k32.2) 2 expts. : 0 5 expts.: 2.11 (k0.76) 66.75 (k10.6) 4.40 (k2.70)
P130
S230
P230
G6PDH
Protein
ONPGH
G6PDH
Protein
ONPGH
G6PDH
Protein
ND
22.15 (f4.60)
40.80 (f17)
ND
17.9 (f6.7)
163.6 (k73.35)
ND
50.80 (f9.6)
ND
-
1 (k0.28)
ND
-
5.35 (f2.55)
ND
-
13.50 3expts.:O ( k 6.25) 1 expt. : 45
ND
18.8 (k7.7)
ND
61.5 (k7.5)
ND
-
0
12 (f2.8) -
ND ND
2expts.:O 0 2 expts.: 14.89 (Traces) 1 expt.: 0 0 2 expts. : 0.85
11.6 ( f 3.5) -
3 expts.: 0 1 expt. : 0.59 1.86 (f 0.93) 0.09 (kO.01)
0
1 expt.: 0 1 expt.: 149.6 2 expts. : 28.85 0.88 (0 to 1.94) 10.3 (k3.00) 0.64 ( k 0 . 15)
ND
0 0
ONPGH 24.7 (k17.6) 0.63 (0.09 to 2.65)
G6PDHb
ZAFIAC
OD 660 nm variationc
2.5
-18.7
(k0.88)
(1.8 to 28)
1.23 (f0.83)
-23.3 (k6.8)
ND
ND
1 expt.: 25.8 ND 3expts.:6 (k4.1) 1 expt. : 0 197 ND 3 expts.: 0 043 ( k 0.029) 67.9 2 expts.: 0 83 (f5.70) 2 expts.: 44.6 ( k 3 ) - 1 expt.: 0 0.157 2 expts.: 3.27 ( f 0.04)
0.48 (k0.28)
+ 89 (k25.7)
TABLE2. Protein, ONPGH, G6PDH distribution in the fractions obtained from cells grown under vigorous agitation in tubes, treated then according to Gilleland et al. (13) s130
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
Control (washed bacteria) Proteina
ONPGHa
G6PDHanb
Protein
ONPGH
P130 G6PDH
% of control
TSB 2 , 8 h (TES 8.6) (3 expts.)
% of control
(3 expts.)
G6PDH
Protein
ONPGH
G6PDH
Protein
OD variation, 660 nm
ONPGH
G6PDHb
73.9 (k48.4) 3.07 (k1.86) 15.7 (k2.3) 0.97 (k0.94)
U/mg
% of control
TSB 2, 15 h (TES 8.6) (7 expts.) (but for G6PDH: only 4 expts.) TSB 2, 15 h (TES 8.6; 0.1)
ONPGH
P230
1 expt.: 0 2 expts.: 67.2 1 expt.: 0 2 expts. : 3.34
U/mg
TSB 2, 8 h (TES 8.6; 0.1)
Protein
S,30
U/mg
% of control
34 (k0.07)
1 expt.: 0 lexpt.: 3 . 8 1 expt.: 16.2
8.4
(3 expts.) (but for G6PDH: only 2 expts.)
U/mg
3 (k0.4)
0.08
BHI, 15 h (TES 8.6)
% of control
1 expt.: 0 1 expt.: 0.08 1 expt.: 0.69 21.2 ( k 16)
(3 expts.) (but for G6PDH: only 2 expts.)
U/mg
.Mean values. bDeterm~nedon a sonicate of bacteria. denotes a decrease, an increase in OD 660 nm. "tandard d e v ~ a t ~ o n . NOTE:ND, not done.
+
25.6 ( t 6.0)
Traces
8.3 (k2.3)
0.85 ( k 0 . 18)
Traces
0.32 (+O. 14)
60
XAF/AC
%'
POINDRON ET A L
TABLE 3. Stimulating effect of heated S,30 on ONPGH activity (TSB 2 grown bacteria, treatment according to Gilleland er 01. (13))
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
ONPGH activityh
Crude S130 (3 expts.)
+ phosphate buffer 1.35 ( k 0 17)'
Heated S,30d (3 expts.) Crude S130
100
+ phosphate buffer
+ heated S130 (3 expts.)
0
0
2 14 ( + 0 36)
I61 ( k 4 4 )
"One volume of each reagent. bMean values. S t a n d a r d deviation. *Crude S130 was heated to 60°C for 30 rnin, a n d slowly cooled to 30'C within 2 11
TABLE 4. Inhibitory effect of CHCI, treatment on ONPGH activity of diverse fractions Crude fraction ONPGH activityh
WF (3 expts.) S130 (3 expts.) S J 0 (1 expt.)
CHC13 treated" ONPGH activity"
Ulnlg
%
Ulmg
%
0 51 (i-0 2.37 ( k 1 . 1 7 ) 2.65
100 100 100
0 043 ( k 0 01) 0 73 ( L o 089) 0 37
8 5 (k2) 56 (k29) 13.9
OOne volume of fraction was mixed with one volu me of CHCI, and shaken with a vortex, 3 times f o ~ 1 min. bMean values.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
PHILLIPPE POINDRON, ALAINBOURGUIGNAT, FRANGOISE SCHEID, A N D YVESLOMBARD Unit~c~rsi~P Loriis PcrslcJrrr, U . E . R . clc,s Sciences Plicrr~irrrcerr~iqrres, Lcrboraroirc,~de Virologic el Micr.oDiologie gPrlPrnle, 3 , r?re de I'Argorine, F-67083 Srrtrsborrrg, C ~ d e . rFrcrnce ,
Accepted February 17, 1977 POINDRON, P., A. BOURGUIGNAT, F. SCHEID. and Y. LOMBARD. 1977. The orthonitrophenylbeta-D-galactoside hydrolase from Pserrdomortcrs nerrrginoscr (0: I 1 serotypes) is a superficial enzyme. Can. J . Microbiol. 23: 798-810. About 95% of the 0 : l l strains of Pserrdornonrrs trerrrgir~oscr was able to hydrolyze orthonitrophenyl-beta-D-galactopyranoside(ONPG), but was unable to use lactose. The ONPG-hydrolyzing enzyme was located essentially in the periplasm, as seen by biochemicaland ultrastructural studies. POINDRON, P., A. BOURGUIGNAT, F. S C H E I D et Y. LOMBARD. 1977. The orthonitrophenylbeta-D-galactoside hydrolase from Psrrrdornoricrs crerrrginosa (0: 11 serotypes) is a superficial enzyme. Can. J. Microbiol. 23: 798-810. Environ 95% des souches 0:ll de Pserrdomonrcs aerr~ginoscr sont capables d'hydrolyser I'onhonitrophenyl-beta-~-galactopyranoside(ONPG), mais ne peuvent utiliserle lactose comme source de carbone. Pour mieux comprendre le r6le de I'enzyme hydrolysant I'ONPG, les auteurs etudient la localisation de I'enzyme dans la bacterie. Celui-ci est situe essentiellement dans le periplasme, comme le montrent les etudes biochimiques et ultrastructurales.
Introduction The microbiologists admit generally that Pseudorno~iasaeruginosa is unable to hydrolyze orthonitrophenyl - beta - D - galactopyranoside (ONPG). Nevertheless a few years ago, Bulow (4) found that some strains of the species were endowed with this property. We were unaware of this observation when we stated during an epidemiological study (22) that about 95% of the 0 : 11 strains (according to Habs (14) and Veron (28)) synthesized such an enzyme. Since our first study which dealt with 240 strains of all serotypes, we extended this observation to a population of 647 strains originating from several hospitals. Since the 0 : 1 1 strains were unable to grow in lninimal medium added with lactose, the questions of the localization and roles of the ONPGhydrolyzing ~noleculearose. We report here a preliminary response to these questions. Materials and Methods Bac~erialSlrains, Media, and Grobvrh Condirions
The D 72/28 strain of our collection was chosen as representative of 0 : I I serotypes, because it possessed the typical characters of the species and rapidly hydrolyzed ONPG (according to Le Minor and Ben Hamida (17)). The strain was grown either in trypticase soja broth
(TSB) (BD MCrieux) or in brain heart infusion (BHI) (Institut Pasteur Production). The choice of the latter medium was circumstantial; we used i t because the results obtained in TSB depended on the batch used. The CaZ+ and Mg2+ concentrations of the media might be of importance to interpret the results; we measured them by atomic absorption spectrophotometry (TSB batch 1, reference not recorded, divalent cations not determined; TSB batch 2, reference No. 251 1, C a Z +: 28 mg/P, Mg2+: 17.6 mg/P; BHI, reference No. 1318, C a Z +: 16.4 mg/P, Mgzf : 7.6 mg/e). In some cases, we used minimal medium (glucose: 0.0139 M , KZHPOJ: 0.4 M, KHzPOA: 0.022 M , MgS04 and CaCI, adjusted at the required concentration). The bacteria were grown at 37"C, either in a 250-ml flask (100ml of medium under mild agitation (90 to and fro movements per min), or in tubes (14 x 140 mm) (5 ml of medium) under vigorous agitation (1 60 to-and-fro movements per minute). When experiments were carried out in minimal medium, sterilizable polypropylene tubes were used. Asscry of EIIZJJI~IPS
ONPG-hydrolase (ONPGH) was assayed at 30°C according to Dean and Smith (11) except for the incubation period which was longer (I8 h). We shall justify this choice in another paper. One unit (U) corresponds to the amount of enzyme that is able to hydrolyze 1 nmol o n ONPG per minute. Glucose-6-phosphate dehydrogenase (G6PDH, E C 1.1.1.49) is a cytoplasmic enzyme. It was assayed according t o D e Moss (12) to check the leakage of cytoplasmic material in the fractions.
POlNDRON ET AL.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
Assay of Proteit~s It was performed according to Lowry et 01. (19). Fractionation of tile Bactet.ia1 Cells Unless otherwise mentioned, all operations were carried at 4'C. In most instances, we treated the bacteria according to the method of Gilleland et a/. (13): 10 ml of appropriate culture was centrifuged at 6SOOg for 20 min. The supernatant was discarded, and the pellet was washed in deionized water, and centrifuged again in the same conditions. The pellet was then resuspended in 0.03 M tris(hydroxymethyl)amino methane (Tris) HCI, pH 8.6, containing I niM ethylenediaminetetraacetic acid (EDTA), and 0.6 M sucrose (N-tris(hydroxymethy1)methyl-2-amino-ethanesulfonic acid (TES) 8.6) (80 ml of TES 8.61250 mg of bacteria in wet weight). In someexperiments we used 0.1 m M EDTA instead of 1 m M (TES 8.6; 0.1). The suspension, called 'complete system,' was gently shaken on a rotative shaker for 1 h at room temperature. Its optical density at 660 nm was measured, and referred to as 100%. The suspension was then centrifuged at 6500g for 20 min. The opalescent supernatant (S,6.5) was centrifuged again at 30 0 0 0 g for 10 min. Proteins and enzymes were assayed in the resulting supernatant (S130) and pellet (P,30). TES 8.6 - treated P. aerugitloso are osmotically fragile. They are called for this reason 'osmoplasts' (13). We checked the osmotic fragility of the osmoplast pellet (P,6.5) as follows: P16.5 was rapidly suspended in deionized water and vigorously shaken with a Vortex shaker for exactly 1 min. The optical density at 660 nm of this suspension was measured immediately, and again after 1 h at room temperature. The osmotic fragility was expressed as a percentage of decrease in optical density, by comparison with the optical density of the complete system set at 100%. The shocked suspension was finally centrifuged at 30 0 0 0 g for 20 min. The supernatant (S230) and the pellet (P,30) were assayed for proteins and enzyme. Because of the effect of TES 8.6 treatment on the cytoplasmic membrane of P. aerr~git~osa, we used also a milder procedure to fractionate the bacteria (Neu and Chou (20)). They were treated exactly as above with the following two modifications. (I) The initial washing of the bacteria was made with 0.03 M TrisNaCI, p H 7.3, instead of deionized water. (11) TES 8.6 was replaced by 0.03 M Tris.HCI, pH 7.3, containing 1 m M EDTA and 0.6 M sucrose. Electron-microscopic Observations Ultratl~inSections The samples were fixed for 2 h, at 4'C, with a mixture formed of one volume of 25% (v/v) glutaraldehyde and four volumes of 0.1 M phosphate buffer, pH 7.4. After thorough washings with 0.1 M phosphate buffer, the samples were treated with a mixture formed of one volume of 2 x (w/v) osmic acid and one volume of 0.2 M phosphate buffer. They were preembedded in buffered agarose, dehydrated, and embedded in Epon Araldite. Sections were made with an O M U 2 Reichert ultramicrotome. They were stained with a mixture o f uranyl acetate and lead citrate. Negative stainings These were made, following standard techniques, with potassium phosphotungstate.
799
Results Preliminary Observatiot~son ONPG H Preliminary experiments showed that the shock fluid S,30 from TSB 1 - grown bacteria, treated according to Gilleland er al. (13), had a good ONPGH activity. We therefore used S,30 to check if ONPG-hydrolyzing property is due to an enzyme. Heating at 100°C for 1 h, or treatment for I h at 37°C with 0.25% buffered trypsin, pH 7.3, destroyed the hydrolytic activity. The ONPG hydrolysis by the 0: l l strains can therefore be ascribed to a thermolabile protein. In whole bacteria, enzyme activity was suppressed by toluene or chloroform; in siru lipids might play a role in the normal functionning of ONPGH. We observed also that the enzyme was not stable at 4°C.
I17j7ueiice of the Groit~tl7Medium on the Bekavio~ir of the Bacterial Cells upon T E S 8.6 Treatment Nossal and Heppel (21) have shown that the selective release of enzymes by osmotic shock takes place under milder conditions with exponentially growing cells than with Eschericl?ia coli in stationary phase. We therefore studied first 15-h-old bacteria, assuming that P. aeruginosa which is very sensitive to the combined action of Tris and EDTA would be less fragile in these conditions; we observed that the results depended both on growth conditions and culture medium. Culture in Flask under Mild Agitation (Table 1 ) Table I shows that protein distribution within the various fractions does not depend upon the nutritional conditions imposed to the bacterial cells. This is not the case for ONPGH activity. The S,30 and P,30 fractions from BHI-grown bacteria contained lower levels of ONPGH activity than those from TSB-grown bacteria. With TSB 1, the S,30 contained 160% of the total ONPGH activity of the whole bacteria taken as control. At first sight this result is in favor of a periplasmic localization for ONPGH. With TSB 2, results were heterogeneous: enzyme activity varied from one experiment t o another. It was always low in the S230 from BHI-grown cells. With all media, little, if any, activity was detectable in the P230. In all series of experiments we found cases where no enzyme activity was detectable in the fractions. Since the protein distribution did not
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
800
CAN.
I. MICROBIOL. VOL. 23. 1977
depend on the nutritional conditions, this apparent lack of activity on O N P G H must be related to qualitative (limiting cofactors) rather than quantitatives factors (amount of proteins). In deionized water, BHI osmoplasts behave unexpectedly. The optical density at 660 nm of the complete system did not decrease as with TSB 1 and TSB 2 osmoplasts, but increased. Since G 6 P D H activity was absent in S130 and in S230 from BHI-grown bacteria, we may conclude that the O N P G H activity detected in these fractions originated from superficial sites of the bacterial cell. The comparison between the sum of the O N P G H activity of the four fractions (CAF) and the activity of the control sample (AC) provided further information. The ratio EAF/ A C was about 2.5 in TSB 1 experiments (7 expts.), 1.2 in TSB 2 experiments (4 expts.), and 0.5 in BHI experiments (3 expts.). The enzyme seems to be 'cryptic' in TSB experiments, and loses its activity in the case of BHI-grown cells. Since in both media, a sonicate of washed bacteria had exactly the same specific activity as washed unbroken control bacteria, we may conclude that the apparent crypticity is not due to inaccessibility of the enzyme in vivo, but to another factor which depends chiefly on the culture medium and the fractionation.
this latter enzyme was present in the corresponding fractions from TSB-grown bacteria.
Influence of Age (Table 2) The results observed with 15-h-old bacteria might reflect the physiological state of bacteria sampled a t the beginning of the stationary phase and, more particularly, the heterogeneity of the bacterial population. We studied thus 8-h-old bacteria, grown in tubes under vigorous agitation. In these conditions, the cells had a higher O N P G H specific activity. T h e protein distribution in the fractions was not affected. However, the age influenced the O N P G H distribution. Now the S,30 and the S230 had two t o seven times higher levels of O N P G H activity. With TES 8.6 - treated cells, the mean ratio EAFIAC was about 1 ; with TES 8.6; 0.1 treated-cells, it was about 1.6.
Stin7ulation or Inhibition of the ONPGH Activity The correlation between the serotype and the presence of O N P G H activity, the superficial localization of this enzyme, its sensitivity t o organic solvents, the heterogeneous results observed in certain conditions, the apparent crypticity, o r the apparent loss of the enzyme activity suggested to us (I) that this enzyme might play a role in lipopolysaccharide (LPS) Culture in Tubes under Vigorous Agitntion biosynthesis and (11) that it required several (Tc1ble 2 ) factors to function normally, probably a thermoThe results were analogous but not exactly stable carbohydrate moiety, non-covalentlyidentical with the previous data. We found the linked lipids, and the O N P G H itself. W e have following differences. (I) When grown in TSB, assumed that each of these factors was critical the whole cells had a three-to-four times higher for the hydrolysis reaction. T E S 8.6 treatment O N P G H specific activity whereas this specific would dissociate more or less the components, activity remained unchanged with BHI-grown whose respective concentrations in the fractions bacteria. (11) The protein distribution was dif- might vary, depending on the age and the culture ferent and reflected a greater sensitivity of the conditions. If this hypothesis is correct, recell to TES 8.6 treatment. (Much more proteins combination experiments between crude fracin S,30 and S,30, greater decrease of the tions on the one hand, and heterologous o r optical density of the TSB osmoplasts in de- homologous fractions treated in various ways o n ionized water, smaller increase with BHI osmo- the other one, must reveal modifications in the plasts.) The replacement of TES 8.6 by TES 8.6; enzyme activity of the crude samples used as 0.1 did not change the results except for the source of enzyme. lower sensitivity of the osmoplasts to the hypoIt was possible to stimulate (Table 3) the tonic shock. (111) The ratios CAF/AC were O N P G H activity in S,30 upon addition of a n always of about 0.5. The O N P G H activity is lost equal volume of a homologous preparation, during the fractionations. heated to 60°C, and slowly cooled to 30°C (such The BHI osmoplasts resisted the hypotonic a treatment increases acceptor activity o f shock; there was no G 6 P D H either in the S130 Snlmonelln LPS by allowing its optimal associao r in the S,30 from BHI-grown cells, whereas tion with phospholipids (24, 25)). The heated
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
TABLE 1. Protein, ONPGH, and G6PDH distribution in the fractions obtained from cells grown under mild agitation in balloon flask, treated then according to Gilleland ef al. (13) Control (washed bacteria) Medium TSB batch 1 (15 h) (7 expts.)
Protein" ONPGH" G6PDH"sb
% of control
100
ND
8.90 (k2.8)
-
0.62d (k0.22)
ND
-
100
100
ND
8 (k3.5)
-
0.47 (k0.07)
ND
-
% of control
100
100
100
U/mg
-
0.67 (f0.06)
0.199 (k0.06)
11.2 (k2.3) -
% of control
U/mg
BHI (15 h) (4 expts.)
Protein
100
U/mg TSB batch 2 (15 h) (4 expts.)
S130
nMean values. *Determined o n a sonicate o f bacteria. Cdenotes a decrease. an increase m the O D 660 nm. dstandard deviation. NOTE:N D , not done.
+
ONPGH 2 expts. : 0 5 expts.: 51 (k32.2) 2 expts. : 0 5 expts.: 2.11 (k0.76) 66.75 (k10.6) 4.40 (k2.70)
P130
S230
P230
G6PDH
Protein
ONPGH
G6PDH
Protein
ONPGH
G6PDH
Protein
ND
22.15 (f4.60)
40.80 (f17)
ND
17.9 (f6.7)
163.6 (k73.35)
ND
50.80 (f9.6)
ND
-
1 (k0.28)
ND
-
5.35 (f2.55)
ND
-
13.50 3expts.:O ( k 6.25) 1 expt. : 45
ND
18.8 (k7.7)
ND
61.5 (k7.5)
ND
-
0
12 (f2.8) -
ND ND
2expts.:O 0 2 expts.: 14.89 (Traces) 1 expt.: 0 0 2 expts. : 0.85
11.6 ( f 3.5) -
3 expts.: 0 1 expt. : 0.59 1.86 (f 0.93) 0.09 (kO.01)
0
1 expt.: 0 1 expt.: 149.6 2 expts. : 28.85 0.88 (0 to 1.94) 10.3 (k3.00) 0.64 ( k 0 . 15)
ND
0 0
ONPGH 24.7 (k17.6) 0.63 (0.09 to 2.65)
G6PDHb
ZAFIAC
OD 660 nm variationc
2.5
-18.7
(k0.88)
(1.8 to 28)
1.23 (f0.83)
-23.3 (k6.8)
ND
ND
1 expt.: 25.8 ND 3expts.:6 (k4.1) 1 expt. : 0 197 ND 3 expts.: 0 043 ( k 0.029) 67.9 2 expts.: 0 83 (f5.70) 2 expts.: 44.6 ( k 3 ) - 1 expt.: 0 0.157 2 expts.: 3.27 ( f 0.04)
0.48 (k0.28)
+ 89 (k25.7)
TABLE2. Protein, ONPGH, G6PDH distribution in the fractions obtained from cells grown under vigorous agitation in tubes, treated then according to Gilleland et al. (13) s130
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
Control (washed bacteria) Proteina
ONPGHa
G6PDHanb
Protein
ONPGH
P130 G6PDH
% of control
TSB 2 , 8 h (TES 8.6) (3 expts.)
% of control
(3 expts.)
G6PDH
Protein
ONPGH
G6PDH
Protein
OD variation, 660 nm
ONPGH
G6PDHb
73.9 (k48.4) 3.07 (k1.86) 15.7 (k2.3) 0.97 (k0.94)
U/mg
% of control
TSB 2, 15 h (TES 8.6) (7 expts.) (but for G6PDH: only 4 expts.) TSB 2, 15 h (TES 8.6; 0.1)
ONPGH
P230
1 expt.: 0 2 expts.: 67.2 1 expt.: 0 2 expts. : 3.34
U/mg
TSB 2, 8 h (TES 8.6; 0.1)
Protein
S,30
U/mg
% of control
34 (k0.07)
1 expt.: 0 lexpt.: 3 . 8 1 expt.: 16.2
8.4
(3 expts.) (but for G6PDH: only 2 expts.)
U/mg
3 (k0.4)
0.08
BHI, 15 h (TES 8.6)
% of control
1 expt.: 0 1 expt.: 0.08 1 expt.: 0.69 21.2 ( k 16)
(3 expts.) (but for G6PDH: only 2 expts.)
U/mg
.Mean values. bDeterm~nedon a sonicate of bacteria. denotes a decrease, an increase in OD 660 nm. "tandard d e v ~ a t ~ o n . NOTE:ND, not done.
+
25.6 ( t 6.0)
Traces
8.3 (k2.3)
0.85 ( k 0 . 18)
Traces
0.32 (+O. 14)
60
XAF/AC
%'
POINDRON ET A L
TABLE 3. Stimulating effect of heated S,30 on ONPGH activity (TSB 2 grown bacteria, treatment according to Gilleland er 01. (13))
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/07/15 For personal use only.
ONPGH activityh
Crude S130 (3 expts.)
+ phosphate buffer 1.35 ( k 0 17)'
Heated S,30d (3 expts.) Crude S130
100
+ phosphate buffer
+ heated S130 (3 expts.)
0
0
2 14 ( + 0 36)
I61 ( k 4 4 )
"One volume of each reagent. bMean values. S t a n d a r d deviation. *Crude S130 was heated to 60°C for 30 rnin, a n d slowly cooled to 30'C within 2 11
TABLE 4. Inhibitory effect of CHCI, treatment on ONPGH activity of diverse fractions Crude fraction ONPGH activityh
WF (3 expts.) S130 (3 expts.) S J 0 (1 expt.)
CHC13 treated" ONPGH activity"
Ulnlg
%
Ulmg
%
0 51 (i-0 2.37 ( k 1 . 1 7 ) 2.65
100 100 100
0 043 ( k 0 01) 0 73 ( L o 089) 0 37
8 5 (k2) 56 (k29) 13.9
OOne volume of fraction was mixed with one volu me of CHCI, and shaken with a vortex, 3 times f o ~ 1 min. bMean values.
