Eur. J. Biochem. 58. 273-282 (1975)
Heterogeneity of the Lipopolysaccharide from Pseudomonas aeruginosa Ian R. CHESTER and Pauline M. MEADOW Biochemistry Department, University College, London
(Received May 21 /July 7, 1975)
Lipopolysaccharide isolated from Pseudomonas aeruginosa PACl and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC 1 and PAClR. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.
The lipopolysaccharide of Pseudornonas aeruginosa PAC1 can be isolated by phenol extraction [l] and the lipid A split off by hydrolysis with 1 % acetic acid at 100°C for 60 min [2]. This releases partially degraded polysaccharide which can be separated by fractionation on Sephadex G-50 into high and lowmolecular-weight fractions of different composition. A mutant defective in rhamnose metabolism lacks the high-molecular-weight fraction [2] and we suggested that this might correspond to a side-chain if the pseudomonad lipopolysaccharide had a structure comparable to that of the Enterobacteriaceae (see [3] for review). Similarly the major low-molecular-weight fraction might be derived from a core polysaccharide common to all strains of P.aeruginosa. Evidence for a common core polysaccharide consisting mainly of glucose, rhamnose, heptose, galactosamine, phosphorus and probably alanine has since been obtained in several different strains of P. aeruginosa [4- 91. There is much more variation in the highmolecular-weight fractions released by acetic acid hydrolysis of the isolated lipopolysaccharide. In a preliminary survey of the chemical composition and structure of lipopolysaccharide isolated from strains of P. aeruginosa of different Habs [lo] serotype, we found the high-molecular-weight fractions were re-
sponsible for the serological specificity of the original lipopolysaccharide and varied markedly in chemical composition [4]. In some strains, particularly those of Habs type 3, several high-molecular-weight fractions were detected, while in others, for example a strain of Habs type 2A, the high-molecular-weight fractions isolated on Sephadex G-75 contained no detectable neutral sugars but retained their antigenic specificity and contained amino compounds. The presence of more than one high-molecular-weight fraction with different compositions in lipopolysaccharide extracted from a single strain suggested that the lipopolysaccharide itself might be heterogeneous. The work described here was undertaken to determine whether there could be heterogeneous lipopolysaccharides in strains of P.aeruginosa and how far their structure could be explained in terms of a common core with strain-specific side-chains.
MATERIALS AND METHODS
Bucterial Strains and Growth
P. aeruginosa PACl (formerly known as 8602 [l I]) was used for most of this work. It belongs to Habs
Pseudomonas Lipopolysaccharide
214
serogroup 3 and carries a lysogenic bacteriophage PS1 (P. H. Clarke, personal communication). A spontaneous mutant resistant to this bacteriophage was isolated and designated PAClR. Its serotype and all other properties tested, except that of sensitivity to PS1, appeared to be unchanged from the parent strain PAC1. Strains P. aeruginosu 2A and 9 were the Habs type strains [9]. Colindale 9 was isolated from clinical material in the Central Public Health Laboratories, Colindale, London. Colindale 9PA was a spontaneous mutant selected by its resistance to bacteriophage 7 [12]. The bacteria were grown at 37 "C from a 1 % inoculum in 1-1 batches in 5-1 flasks shaken at 37°C or in a 1000-1 fermenter with an aeration of 300 l/min. The medium used was either nutrient broth (Oxoid no. 2) or minimal medium [13] supplemented with sodium succinate (0.5 %). When they reached the stationary phase of growth the bacteria were harvested by centrifugation at 15000 x g for 30 min, washed twice with distilled water and their walls prepared after disruption in a French pressure cell as previously described [4].
Isolation and Degradation of' Lipopolysaccharide Lipopolysaccharide was normally isolated from defatted walls by extraction with 45 "/, aqueous phenol [l]. The aqueous phases were centrifuged to remove contaminating peptidoglycan, dialysed and freezedried as described by Key et al. [14]; yield approx. 100 mg/g walls. Where specified the petroleum ether/ chloroform/phenol method [15] was also used. Lipid A was removed from the isolated lipopolysaccharide (25 mg) by hydrolysis with acetic acid [2]. The lipid was extracted with chloroform and the watersoluble products (partially degraded polysaccharides) freeze-dried. They were separated by elution from a column (3 x 55 cm) of Sephadex G-75 with pyridinel acetate buffcr (0.05 M, pH 5.4) [6]. Fractions (4 ml) were analysed for neutral sugar content by the phenol/ sulphuric acid method [ 171. For alkaline de-0-acylation lipopolysaccharide (50 mg) was suspended in 50 ml99 aqueous ethanol (0.1 N in NaOH) and incubated for 45 min at 37 "C with occasional shaking. The resulting product was neutralised with hydrochloric acid (2 N), evaporated to dryness at 4'C by rotary evaporation and the residue dissolved in about 3 ml of pyridine/acetate buffer (0.05 M, pH 5.4). The solution was centrifuged (I5000 x g , 5 min) to remove any insoluble material and the supernatant fluid filtered through sintered glass (no. 1 porosity). It was applied to a column (3 x 55 cm) of Sephadex G-200, eluted with pyridine/ acetate buffer, and fractions analysed with phenol/ sulphuric acid as before.
Anulytical Methods Methods used for the determination of total phosphorus and glucose and for the detection and estimation of amino compounds by automatic analysis were all as described previously [4]. No correction was made for the destruction or slow release of components during hydrolysis. The compound previously designated U4 [4] was identified by comparison with an authentic sample as fucosamine. The amounts of the unknown amino compounds were calculated assuming a colour yield of 1 and a molecular weight equivalent to that of glucosamine. 2-Keto-3-deoxyoctonic acid, rhamnose, fatty acids and heptose were all determined as described by Fensom and Gray [18]. Amounts of heptose were calculated as L-glycero-D-mannoheptose using the standard values obtained by Osborn [19]. RESULTS The lipopolysaccharide analysed previously [2] had been isolated from whole organisms of P. aeruginosa PACl grown in 1-1 batches in minimal medium with citrate as carbon source. Lipopolysaccharide isolated from walls of this strain grown in 1-1 batches in nutrient broth was very similar both in yield and composition (Table 1, batch 1). The main sugar components were glucose, rhamnose, heptose and 2-keto3-deoxy-octonic acid. The amino compounds present, glucosamine, glucosamine phosphate, galactosamine, fucosamine and two unidentified amino compounds designated U2 and U3, had all previously been detected in lipopolysaccharide isolated from PACl and another Habs serotype 3 strain [4]. In order to obtain enough material to analyse in detail, we grew larger batches in a 1000-1 fermenter and, to prevent induction of the lysogenic bacteriophage PS1, used a spontaneous mutant resistant to it, PAClR. The lipopolysaccharide prepared from walls of this mutant grown in 1-1batches in nutrient broth and minimal medium with succinate as carbon source (Table 1, batches 2 and 3) had very similar chemical composition to that of PACl . Similarly lipopolysaccharide isolated from PACl R grown in 500 1 of nutrient broth in the fermenter had the same composition (Table 1, batch 4). However when a 500-1 batch was grown in minimal medium with succinate as carbon source the lipopolysaccharide isolated was quite different (batch 5) and will be described as abnormal lipopolysaccharide. It was deficient in heptose, glucose, alanine, galactosamine and glucosamine phosphate as compared with the other preparations analysed. It also contained particularly large amounts of a material previously detected by automatic amino compound analysis in hydrolysates of normal polysaccharide from PACl and another strain of Habs type 3 serotype [4]. This had a high
I. R. Chester and P. M. Meadow
215
Table 1. Elfect of growth conditions on the composition of lipopolysaccharide from P.aeruginosa PACl and PACIR Results are expressed as percentages of lipopolysaccharides ~
Component
Glucose Rhamnose Heptose Phosphorus Glucosamine phosphate A I an i n e Glucosamine Galactosamine U2" u3 a Fucosamine 2-Keto-3-deoxyoctonic acid "
Amount present in batch
1 (PACl in 1-1 nutrient)
2 (PAClR in 1-1 nutrient)
3 (PACIR in 1-1 succinate)
4 (PACIR in 500-1 nutrient)
5 (PAClR in 500.1 succinate)
10.2 9.6 4.2 4.6 1.6 1.1 6.8 1.5 1.8 0.1 trace 3.4
8.4 11.6 5.6 4.8 1.5 1.2 8.0 1.8 1.6 0.2 0.2 2.4
9.9 11.9 5.2 4.4 1.8 1.2 7.8 1.6 1.4 0.2 0.1 2.4
12.8 10.9 4.4 4.5 2.6 1.3 8.6 1.6 1.6 0.4 trace 2.9
0.3 15.9 0.4 1.o 0.4 0.4 13.2 0.5 2.8
0.6 trace 0.9
Unidentified amino compounds [4].
absorbance at 440 nm after ninhydrin treatment and was basic, being eluted just after galactosamine. It has not yet been identified. The low heptose content of the abnormal lipopolysaccharide suggested that if might have less than the normal amount of lipid A. When it was treated for 60 min with 1 yo acetic acid which precipitates lipid A from normal lipopolysaccharide of PACl R and PAC1, there was no precipitate, although prolonged hydrolysis caused slight cloudiness. Treatment with 0.1 N HC1 which splits lipid A from some pseudomonad lipopolysaccharides [4] also failed to release appreciable amounts of lipidA from the abnormal lipopolysaccharide. However by increasing the amount of abnormal lipopolysaccharide three-fold (100 mg) and hydrolysing with 1 % acetic acid at 100°C for 150 min, sufficient lipid A was released for analysis. The fatty acids present were those previously identified in lipid A from PACl [20] but the amount (6.8 %) was lower than in the normal lipopolysaccharide (20.9 "/,). One possible explanation of the unusual composition of the abnormal lipopolysaccharide would be that under certain conditions a lipopolysaccharide was produced which was not readily extracted by the hot phenol method used. The rough lipopolysaccharidcs of the Enterobacteriaceae require a petroleum ether/ chloroform/phenol mixture for extraction [151. However walls of P. aeruginosa from which either normal or abnormal lipopolysaccharide had been extracted by aqueous phenol contained no detectable heptose or rhamnose which might indicate unextracted lipopolysaccharide. Furthermore, extraction of the dried walls with petroleum ether/chloroform/phenol only 1 of the lipopolysaccharide obtained by aqueous
phenol, although lipopolysaccharide isolated using the aqueous phenol method was soluble in petroleum ether/chloroform/phenol. Neither heptose nor rhamnose were detected in the phenol phase or insoluble residue after extraction of walls with aqueous phenol. There was thus no evidence for additional heptosecontaining or rhamnose-containing polymers in the walls other than those extracted into the aqueous phenol. A number of other growth conditions have been studied but abnormal lipopolysaccharide has been isolated only from cultures grown in the 1000-I-fermenter with succinate or lactate as carbon source. It has not so far been possible to determine the factors controlling abnormal lipopolysaccharide production. Acetic Acid Degradation of PACIR Lipopolysaccharide
The differences between the normal and abnormal PACl R lipopolysaccharides were even more striking after hydrolysis with 1 acetic acid and fractionation of the partially degraded polysaccharides on Sephadex G-75. The normal lipopolysaccharide like that from the Habs type 3 strain studied previously [4] was degraded into two major high-molecular-weight polysaccharide fractions distinct from the low-molecularweight fractions (Fig. 1A). 3-Keto-3-deoxyoctonic acid and phosphate were released as low-molecularweight solutes eluted after fraction L. Although acetic acid treatment of the abnormal lipopolysaccharidc released little lipid A, polysaccharides were released and could be fractionated into high and low-molccularweight components (Fig.1B). Most of the carbohydrate-containing material was eluted as a single
Pseudomanas Lipopolysaccharide
216
l.*IB
1.2 -
aH
A 1
.o -
1
:0.8
L
0.8 -
0.6 -
L
5
a
5
9 0.4 0.2
-
-
9g 0.6
8 * +
.o
0
2
0
0.4-
0.2
160
80
240
-
0' 0
320
i I
80
Eluate (rnl)
I
1
I
160 240 Eluate (ml)
320
Fig. 1, Fractionation on Seplzadex G-75 of pol.vsu(;charide releused by aci>tic trcatment of lipopolysacchuride Jrom P. aeruginosa PAC1 R . Bacteria were grown in 500-1 batches in (A) nutrient broth (normal) and (B) minimal medium with succinate as carbon source (abnormal). Lipopolysaccharide was extracted, purified and hydrolysed with 1% acetic acid as described in Methods. The partially degraded polysaccharidc was eluted from a column (3 x 55 cm) of Sephadex G-75 with pyridine acetic acid buffer pH 5.4.Fractions (4 ml) were analysed for total carbohydrate by the phenol/sulphuric acid method [17]
Table 2. Composition 0f;fractions obtained qfiw acetic acid hydrolysis of normal and uhnormal lipopolysaccharide of P. aeruginosa PAC I R The fractions analysed are shown in Fig. 1 Component
Amount present in high-molecular-weight fractions normal
Glucose Rhamnose Heptosc Phosphorus Glucosaminc phosphate Alanine Glucosamine Galactosamine u2 u3 Fucosamine
low-molecular-weight fractions abnormal
normal
abnormal
H1
H2
aH
L
aL
0.8 18.8 0 0.4 0 0.2 11.1 trace 3.4 0.6 0.6
9.7 28.9 0 0.5 0 0.9 2.1 0.7 0.5 trace trace
1.2 22.2 0 0.2
33.5 6.2 8.4 3.0 0.2 1.6 0.4 2.5 0.2 0.9 trace
16.8 7.2 trace 1.8 0 0.9 trace 1.2 trace 0 0
high-molecular-weight peak (aH) with smaller amounts of lower-molecular-weight material (aL). The major high-molecular-weight fractions (H1 and aH) from the normal and abnormal lipopolysaccharides contained most of the rhamnose glucosamine and fucosamine and unknown amino compounds U2 and U3 of the original lipopolysaccharides (Table 2). The low-molecular-weight fractions contained most of the glucose, heptose, alanine and galactosamine. That from the normal lipopoly-
0
0.3 16.7 trace 4.9 trace 0.8
saccharide (L) contained appreciable amounts of heptose, glucosamine and glucosamine phosphate, but these compounds were present in very much smaller amounts in fraction aL from the abnormal lipopolysaccharide (Table 2). The identifiable components in Table 2 account for only 28-56';/, of the polysaccharide fractions. The unidentified 440-nm absorbing materials present in particularly large amounts in the abnormal lipopolysaccharide were the only other compounds found.
I. R. Chester and P. M. Meadow
211
0.8 r A aN3
0.6
-
0.4
-
a
E 0.6
8-
.J c
m
c
m
a,
e
e 5:
9
0.4
c
0.2
8
-
n -0
I
80
I
I
I
160 240 Eluate (ml)
320
0.2
0
0
80
160 240 Eluate (ml)
320
Fig. 2. Fractionation on Sephadex G-200 of polysaccharide obtained by de-0-acylation bitith ethanolic alkali of lipopolysaccharide ,from P. aeruginosa P A C I R . Bacteria were grown and lipopolysaccharide extracted as described under Fig. 1. Lipopolysaccharide was treated with ethanolic alkali and fractionated on a column of Sephadex (3-200 as described in Methods. Fractions were analyscd for total carbohydrate
Alkali Degradation of PAC1 Lipopolysaccharide If the normal lipopolysaccharide consisted of more than one type of molecule, then the polysaccharide fractions H1 and H2 obtained by acetic acid hydrolysis might have been derived from different lipopolysaccharides one of which, that yielding H2, was not made under the conditions producing abnormal lipopolysaccharide. The isolated lipopolysaccharides were therefore treated with ethanolic alkali before fractionation. This treatment removes 0-acylated fatty acids from the lipid A [21] thus reducing non-polar interactions between lipopolysaccharide components and facilitating their separation. It was hoped that the mild conditions would not degrade the lipopolysaccharides further. Separation of the products of alkaline degradation on Sephadex G-200 gave several fractions from both the normal and abnormal lipopolysaccharides (Fig. 2 A and B). The lowest-molecular-weight fraction of the normal lipopolysaccharide (N4) which accounted for about half the material applied, was completely absent from the abnormal lipopolysaccharide. This was perhaps to be expected since the more drastic acetic acid treatment had also released very little low-molecular-weight material. Analysis of the fractions obtained from the normal lipopolysaccharide showed that they all contained those components concentrated in the low-molecularweight fractions produced by acetic acid degradation ; that is glucose, heptose, alanine, galactosamine and phosphate (Table 3). This suggested that the normal lipopolysaccharide might consist of 3 or 4 types of macromolecules each of which contained the same core fraction with additional high-molecular-weight fractions attached. If so then N4 might be most closely related LO the basic molecule and might correspond to the rough core of the Enterobactcriaceae. Hydrolysis of N4 with acetic acid followed by elution from Sephadex G-75 yielded a polysaccharide whose elution properties and composition were indistin-
Table 3 . Compositions of fractions obtained by elution fiom Sephadex G-200 of material released by ethanolic alkali de-0-acylution of normal 1ipopol.vsaccharide of PAClR Fractions are those shown in Fig. 2 A Component
Amount in fraction N1
N2
N3
N4
0.4 20.5 1.1 1.3 0.7 0.4 12.9 0.6 3.2 0.6 trace 3.3
1.9 19.9
16.9 5.1 5.3 5.7 2.9 2.4 6.2 3.6 0.2 0.1 trace
o/ /"
Glucose Rhamnose Heptose Phosphorus Glucosamine phosphate Alanine Glucosamine Galactosamine u2 u3 Fucosamine 2-Keto-3-deoxyoctonate
6.6 11.4 2.3 2.4 1.9 1.4 11.2 1.9 1.9 0.3 trace 1.7
2.1 1.8
1.3 0.8 4.9
1.4 0.8 0.2 trace 1.8
4.3
guishable from fraction L prepared by acetic acid hydrolysis of the intact lipopolysaccharide. Both N4 and L contained relatively large amounts of glucose suggesting that they might be contaminated with a glucose polysaccharide. However, although fraction L could be further separated by Biogel P 4 into two fractions, both contained glucose linked to the other sugar components (Chester and Meadow, unpublished). It seems unlikely therefore that there is much chemical heterogeneity in the common core polysaccharide. Although fractions N1, N2 and N3 all appeared to contain the same core polysaccharide found in N4 and L, the amounts of polysaccharide common to each fraction must vary since the proportions of the core components varied. These fractions also contained those substances previously found in thc acetic acid degradation products H1 and H2,
278
Pseudomonas Lipopolysaccharide
Eluate (ml)
Eluate (ml)
Fig. 3. Fractionalion q/ purtiully degruded lipop(~ly.~ac~l~uride Jrom P. aeruyinosa 2 A . (A) Acid-hydrolysed products separated on Sephadex G-75. (B) Hydrolysed by ethanolic alkali and separated on Sephadex G-200. Fractions were analysed for total carbohydrate
that is rhamnose, glucosamine, U2, U 3 and fucosamine, U2, U3 and fucosamine. Hydrolysis of N1, N2 and N 3 with 1 "/, acetic acid and separation of the polysaccharides released on Sephadex G-75 confirmed their relationship to H1, H2 and L. N1 and N2 both yielded a major component with identical elution properties to H1 together with smaller amounts of components behaving as H2 and L. N 3 gave mainly material resembling H2 with smaller amounts of L but the amounts of material were too small to analyse. The fractions isolated after de-0-acylation of the abnormal lipopolysaccharide (aN1, aN2 and aN3) (Fig. 2 B) contained the high levels of rhamnose, glucosainine and unknown amino compound U2 characteristic of the acetic acid fraction a H from the same material. The compositions of all three fractions were similar to N 1 and N2 obtained from the normal lipopolysaccharide (Table 4). The presence of appreciable amounts of glucose may suggest a closer resemblance t o N1 than to N2. However the amounts of alanine and glucosamine in aN1, aN2 and aN3 were much less than in N 1 and N 2 and heptose was not detected. This suggests that the amount of core polysaccharide was very much reduced in each of the fractions obtained from the abnormal lipopolysaccharide, as had been indicated by the acetic acid degradation products. Lipopolysaccharide,from Other Strains of P . aeruginosa
The lipopolysaccharides of the Habs type 3 strains to which PAS1 belongs appear to be the most complex of all the strains examined as judged by their hydrolysis products [4]. It was therefore not surprising that alkaline degradation confirmed the complexity of
Table 4. Composition q[frartions obtained by elution from st pi rude.^ G-200 of material released by ethanolic alkali de-0-acylation qf ' abnormal lipopolysaccharide of PAClR Fractions are those shown in Fig.2B Cornponcnt
Amount in fraction aN1 I,
"Y'
Glucose Rhamnose Heptose Alanine Glucosamine Galactosamine u2 u3 Fucosdmine
aN2
aN3
1.3 16.1 0 kdCe 13.1 trace 2.1 2.5 trace
2.6 14.6 0 0.3 12.0 trace 1.8 0.3 trace
,
3.3 8.1 0 0.4 5.1 0 1.o 0 kdCe
PAC1 lipopolysaccharide. It was interesting to see whether there was any evidence for heterogeneity in strains whose lipopolysaccharides yielded fewer products after acid hydrolysis. One of the simplest elution profiles was obtained after acid hydrolysis of lipopolysaccharide from type 2A. Separation of the products on Sephadex G-75 yielded a single low-molecularweight peak (2AL) containing all the neutral sugars of the intact lipopolysaccharide (Fig. 3A). The only material detected in the high-molecular-weight fractions (2AH) were amino compounds. De-0-acylation of the 2A lipopolysaccharide followed by separation of the products on Sephadex G-200 also produced a much simpler pattern than had been found with PAC1 lipopolysaccharide (Fig. 3B). Most of the neutral
I. R. Chester and P. M. Meadow
219 1.2
0 ColSPAN3 1
.o
A
C
9N3
COl9 N2
a
0.8
-3
c
a,
c" m
0.6
5
f \
COlSPANl
3
c
8
p
0.4
0.4
0
P 0.2
80
160
240
320
0
0.2
60
Eluate (ml)
n 160 Eluate ( m l )
240
320
0
80
160
240,
320
Eluate ( m i )
Fig. 4. Fruc,rionation on Sephudu G-200 of partially degraded lipopolysucchuride ,from P. aeruginosa Colindule 9 ( A ) , Colindale YPA ( B ) unii Hubs type Y ( C ) . Lipopolysaccharide was treated with ethanolic alkali and separated on Sephadex G-200 as described in Methods. Fractions were analyscd for total carbohydrate
Table 5 . Di.stributionof amino compounds between fractions separated after arid and alkaline degradation of type 2A lipopolysaccharide The fractions andlysed are those shown in Fig.3. Results are expressed as percentages of the total detected Component
Amount in fraction high-molecularweight
low-molecularweight
2AH
2AL
2AN2
0 YY.8 0
91.1 87.9 86.9
100 5.6 53.4
XY.0
2AN1
0
Glucosaminc phosphate 0 0.2 Alanine Ghcosdmine 0 Galactosaminc < 0.1 94.4 u3 Fucosamine 46.6
8.3 12.1 13.1 11.0 100 47.8
0 52.2
sugars were eluted as a single peak (2AN2) with elution properties and composition very similar to that obtained from PACl (N4, Fig. 1A). The highmolecular-weight fraction (2AN 1) contained small but significant amounts of glucose, heptose, alanine and galactosamine thought to be characteristic of the core polysaccharide. The distribution of the amino compounds between high and low-molecular-weight fractions was very similar following either acid or alkaline degradation except for the loss of glucosamine from the acid fractions. Presumably glucosamine forms part of the lipid A removed by acid hydrolysis. The alanine and galactosamine were concentrated in the low-molecular-weight fractions, U 3 was mainly in the high-molecular-weight fractions and fucosamine
was equally distributed between the two (Table 5). These results suggest that in type 2A as in PAC1 lipopolysaccharide there is more than one structure. The one with lower molecular weight gives rise to 2AL or 2AN2 after acid or alkali treatment. The other contains mainly amino compounds with a smaller amount of the polysaccharide similar if not identical to that of lower molecular weight. Lipopolysaccharides from other strains of P. aeruginosa were also de-0-acylated by treatment with ethanolic alkali and their degradation products separated by elution from Sephadex G-200 (Fig. 4). Each yielded a major component behaving as N4 from PACl together with smaller amounts of highermolecular-weight materials. Those which had yielded complex elution profiles after acid hydrolysis [4] gave several components after alkaline hydrolysis while those with simpler acid hydrolysis products yielded few. Lipopolysaccharide from Habs type 9 which had proved resistant to acetic acid hydrolysis was readily split by ethanolic alkali into at least three components (Fig. 4C). The major product ofthe de-0-acylated lipopolysaccharide from each of the strains was very similar both in elution properties and chemical composition (Table 6). The only differences detected were in the amounts and character of the minor amino compounds. These amino compounds were the same as those previously detected in acid hydrolysed lipopolysaccharide and thought to be characteristic of the strain and possibly the serotype [4]. The other de-0acylation products all contained the common core components as well as the amino compounds detected in the high-molecular-weight fractions obtained by acid hydrolysis of the same lipopolysaccharide.
280
Pseudomonas Lipopolysaccharide
Table 6. Composition of major fractions separated on Sephadex G-200from de-O-acylated lipopolysaccharides of different strains ofP. aeruginosa The fractions analysed are shown in Fig. 2A, 3 A and 4. The amino compounds U1,2,3,5,6,7 are those previously described in these strains [4] Component
Amount in strain (serotype, fraction) .
Glucose Khamnose Heptose Glucosamine phosphate Alanine Glucosamine Galactosaminc
u1 u2 u3 Fucosamine
~
~~~
PAC 1R (3, N4)
2A (2A, 2AN2)
Colindalc 9 (6, Co19N2)
C,olindale 9PA (not typable, Col9PAN3)
9
16.9 5.1 5.3 2.9 2.4 6.2 3.6 0 0.2
14.6 3.0 5.5 2.7 2.4 4.9 3.4 0 0 trace 0.4 0
15.6 6.9 4.4 1.7 2.0 4.1 2.9 0.3 1.6 1.9 3.4 2.7
19.5 4.6 6.1 2.3 2.2 4.6 3.6 tracc 0.6 0.3 1.8 0.9
15.5 5.3 4.7 2.4 1.9 3.9 2.5
0.1
trace 0
DISCUSSION The phenol-extracted lipopolysaccharides of several strains of P. aeruginosa have now been analysed in a number of laboratories and their overall composition found to be very similar [2,4-6,8,9,22]. They can be partially degraded by acetic acid and separated into fractions of differing molecular weight. The lowermolecular-weight fractions appear similar if not identical in all strains and may represent a common core polysaccharide similar to that found in the Enterobacteriaceae. The high-molecular-weight fractions varied between strains and some at least of this variation may depend on their O-serotypes. In strain PACl and its bacteriophage-resistant mutant PAClR the high-molecular-weight fractions separated after acetic acid hydrolysis contained both neutral sugars (mainly rhamnose and glucose) and most of the glucosamine and unidentified amino compound U2 of the complete lipopolysaccharide. Wilkinson and Galbraith [8] have found an amino compound X with similar properties to U2 in high-molecular-weight fractions of lipopolysaccharides from other strains of P. aeruginosa including one of Habs serotype 3. They suggest it may be an aminohexuronic acid. Most of these minor amino components of the lipopolysaccharides seem to be concentrated in the highermolecular-weight fractions obtained after both acid and alkaline degradation of the strains studied here. Aminosugar-rich fractions have also been obtained by deoxycholate treatment of P. aeruginosa P14 (serotype 1) [6]. Again these fractions were of higher molecular weight than the fractions rich in neutral sugars. The role of the high-molecular-weight aminosugarrich fractions of the lipopolysaccharides in the pheno-
(9, 9N3)
0 0
3.3 2.9 0
type of thePseudomonads is difficult to assess. Mutants defective in these fractions appear to have lost their serological specificity [7] (and Koval and Meadow, unpublished) and isolated high-molecular-weight fractions react with homologous antisera [4,8]. However there is not yet enough evidence to link specific amino compounds or groups of compounds conclusively with Habs serotype. On the other hand Suzuki has suggested that the presence of quinovosamine in lipopolysaccharides of P. aeruginosa may confer sensitivity to pyocins A and S. A pyocin-resistant mutant lacks quinovosamine which occurs solely in the highermolecular-weight fractions of the lipopolysaccharide [22]. In the few available mutants of PACl which are defective in high-molecular-weight fractions of the lipopolysaccharide we have so far been unable to find any common phenotype. The overproduction of high-molecular-weight fractions of the lipopolysaccharide of PAClR under certain growth conditions is difficult to interpret particularly as we have been unable to define the cause. The cultures producing abnormal lipopolysaccharide had the same growth characteristics and produced the same final yield as those producing the normal lipopolysaccharide. The bacteria had normal morphology and were indistinguishable from the wild type by any of the biochemical or genetic tests used. The production of abnormal lipopolysaccharide appears to be phenotypic rather than genotypic. It may simply be an extreme example of the heterogeneity normally apparent in pseudomonas lipopolysaccharide and result from changes in the control of lipopolysaccharide synthesis. However although the highmolecular-weight fractions obtained from alkaline degradation of the abnormal lipopolysaccharide were
1. R. Chester and P. M. Meadow
similar to those from the normal there were some differences. The content of core pol ysaccharide was much less than in the normal fractions and lacked the heptose found in the latter. Changes in heptose content of dried walls of Aerohacter aerogenes were shown by Ellwood and Tempest to be related to growth conditions [23]. The lowest heptose contents were obtained in glycerol-limited cultures using low dilution rates. In preliminary experiments using similar conditions we have so far been unable to produce similar results in P.aeruginosu PACl and we do not know what causes production of abnormal lipopolysaccharide with low heptose content under some circumstances in a 1000-1fermenter. In the Enterobacteriaceae heptose is part of the core to which the lipid A and common and antigen-specific polysaccharides are attached [3] and a similar structure has been suggested for P. aerughosa 1999 [9]. It is difficult to see how the abnormal lipopolysaccharide of PACl could be reconciled with this type of structure and more detailed chemical analysis will be required. One of the unusual features of the pseudomonad lipopolysaccharide is the relatively low (16 - 24 o/,) carbohydrate content [4,8]. Even in the isolated polysaccharide fractions we found that only 24- 45 % of the dry weight could be accounted for as carbohydrate. This is near the range (32- 50 %) found by Wilkinson and Galbraith in similar fractions from other strains of pseudomonas [8]. Our analyses did not include ethanolamine, known to form part of the intact lipopolysaccharide [ 5 ] , nor the 440 nm absorbing materials detected by automatic amino compound analysis. Furthermore no correction was made for degradation during hydrolysis. Some of the phosphate is acidlabile [ 5 ] and if U2 is an aminohexuronic acid its content could have been seriously underestimated. Nevertheless a substantial part of the lipopolysaccharide and of its polysaccharide fractions has not been identified particularly in the abnormal lipopolysaccharide from PAC1. We have no evidence as to what the components might be. Both acid and alkaline degradation products of the lipopolysaccharides studied support the view that all strains of P.aeruginosa contain a common core polysaccharide. Specific amino compounds are attached to this in amounts varying from one strain to another and the proportion of the two parts may also vary with physiological conditions. In addition most strains appear to contain other lipopolysaccharides consisting of those components previously detected in the high-molecular-weight acid-degradation products attached to smaller lengths of core polysaccharide. In some strains, particularly PACl and Habs type 9 there was evidence for more than one type of highmolecular-weight fraction possibly corresponding to more than one type of side chain.
28 1
On the lirnitcd evidence available it is difficult to construct a picture of the lipopolysaccharide of P . aeruginosa. The similar fatty acid, phosphate and 2-keto-3-deoxyoctonate contents of the lipopolysaccharides isolated from several strains of P . cieruginosu [4,8] suggest a common lipid A component and the yield of core (L) fractions is remarkably constant [4]. The major alkaline degradation products for different serotypes were very similar except for the specific amino compounds. It is possible that the structure consists of lipid A linked through 2-keto3-deoxyoctonate to the core polysaccharide. To some but not all of these molecules additional side chains could be attached and there may be variation in the numbers of core repeating units. In PACl the normal lipopolysaccharide may consist of at least three types of molecule, one of which contains lipid A linked to the core polysaccharide, a second has the H1 polysaccharide added to this basic molecule while the third has the H2 polysaccharide linked to the core and thence to lipid A. The only lipopolysaccharide-defective mutants we have so far been able to isolate from PACl appear to have lost both the H1 and H2 polysaccharides in a single step (S. Koval and P. M. Meadow, unpublished results). This could mean that H1 and H2 are part of the same lipopolysaccharide molecule, but could also result from a defect in the core. Until more is known of their structure it is not possible to distinguish between the alternatives. We are grateful to the Medical Research Council for a project grant which allowed us to undertake this work. We also thank Mr P. D. Laverack who carried out the automatic amino compound analyses and Mr C. J. Hubball and Mr P. Wells for excellent technical assislance.
REFERENCES 1. Westphal, 0. & Jann, 0. (1965) Meth0d.s Carhohyd Chenz. 5, 83-91. 2. Fensom, A. H. & Meadow, P. M. (1970) FEBS Lert. 9,81- 84. 3. Luderitz, 0..Westphal, 0..Staub, A. M. & Nikaido, H . (1971) Microbial Toxins IV, p. 145, Academic Press, London. 4. Chester, I. R., Meadow, P. M. & Pitt, T. L. (1973) J . Gen. Microhiol. 78, 305-318. 5. Drewry, D. T., Lomax, J. A,, Gray, G. W. & Wilkinson, S.G. (1973) Biochem. J. 133,563 - 512. 6. Ikeda, K. & Egani, F. (1973) J . Gen. Appl. Microbial. 19. 115- 128. 7. Meadow, P. M. (1975) Genetics and Biochemistry of’ Pseudomonas, p. 67, John Wiley & Sons, Chichester. 8. Wilkinson, S. G. & Galbraith, L. (1975) Eur. J . Biochem. 52, 331 -343. 9. Drewry, D. T., Symes, K. C., Gray, G. W. & Wilkinson, S.G . (1975) Biochem. J. 149, 93-106. 10. Habs, I. (1957) Zentralbl. Bakteriol. Parasitenkd. Injektionskr. Hyg. Abt. I Orig. 144, 218-228. 11. Brown, P. R. & Clarke, P. H. (1972) J . Gen. Microhiol. 70, 287-299. 12. Martin, D. R. (1971) Ph.D. Thesis, University of London.
282 13. Clarkson, C. E. & Meadow, P. M. (1971) J. Gen. Microhiol. 66,161 - 169. 14. Key, B. A,, Gray, G. W. & Wilkinson, S. G. (1970) Biochem. J . 120, 55Y - 566. 15. Galanos, C., Luderitz, 0. & Westphal, 0. (1969) Eur. J . Biochem. 9,245- 249. 16. Schmidt, G., Jann, B. & Jann, K. (1969) Eur. J . Biochem. 10, 501 -510. 17. Dubois, M., Gilles, K. A,, Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28,350-356.
I. R. Chester and P. M. Meadow: Pseudomonas Lipopolysaccharide 18. Fensom,A. H. & Gray, G . W . (1969) Biochern. J. 114, 185196. 19. Osborn, M. J. (1963) Proc. Nut1 Acad. Sci. U.S.A. 50, 499506. 20. Hancock, I. C., Humphreys, G. 0. & Meadow, P. M. (1970) Biochim. Biophys. Acla, 202, 389-391. 21. Kasai, N. (1966) Ann. N.Y. Acad. Sci. 133, 486-507. 22. Suzuki, N. (1974) FEBS Lett. 48, 301 -305. 23. Ellwood, D. C. & Tempest, D. W. (1972) Adv. Microh. Physiol. 7 , 83- 117.
I. R. Chester, Fermentation Development Laboratory, Beecham Pharmaceuticals, Clarendon Road, Worthing, Sussex, Great Britain P. M.Meadow, Department of Biochemistry, University College London, Gower Street, London WClE 6BT, Great Britain
Heterogeneity of the Lipopolysaccharide from Pseudomonas aeruginosa Ian R. CHESTER and Pauline M. MEADOW Biochemistry Department, University College, London
(Received May 21 /July 7, 1975)
Lipopolysaccharide isolated from Pseudomonas aeruginosa PACl and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC 1 and PAClR. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.
The lipopolysaccharide of Pseudornonas aeruginosa PAC1 can be isolated by phenol extraction [l] and the lipid A split off by hydrolysis with 1 % acetic acid at 100°C for 60 min [2]. This releases partially degraded polysaccharide which can be separated by fractionation on Sephadex G-50 into high and lowmolecular-weight fractions of different composition. A mutant defective in rhamnose metabolism lacks the high-molecular-weight fraction [2] and we suggested that this might correspond to a side-chain if the pseudomonad lipopolysaccharide had a structure comparable to that of the Enterobacteriaceae (see [3] for review). Similarly the major low-molecular-weight fraction might be derived from a core polysaccharide common to all strains of P.aeruginosa. Evidence for a common core polysaccharide consisting mainly of glucose, rhamnose, heptose, galactosamine, phosphorus and probably alanine has since been obtained in several different strains of P. aeruginosa [4- 91. There is much more variation in the highmolecular-weight fractions released by acetic acid hydrolysis of the isolated lipopolysaccharide. In a preliminary survey of the chemical composition and structure of lipopolysaccharide isolated from strains of P. aeruginosa of different Habs [lo] serotype, we found the high-molecular-weight fractions were re-
sponsible for the serological specificity of the original lipopolysaccharide and varied markedly in chemical composition [4]. In some strains, particularly those of Habs type 3, several high-molecular-weight fractions were detected, while in others, for example a strain of Habs type 2A, the high-molecular-weight fractions isolated on Sephadex G-75 contained no detectable neutral sugars but retained their antigenic specificity and contained amino compounds. The presence of more than one high-molecular-weight fraction with different compositions in lipopolysaccharide extracted from a single strain suggested that the lipopolysaccharide itself might be heterogeneous. The work described here was undertaken to determine whether there could be heterogeneous lipopolysaccharides in strains of P.aeruginosa and how far their structure could be explained in terms of a common core with strain-specific side-chains.
MATERIALS AND METHODS
Bucterial Strains and Growth
P. aeruginosa PACl (formerly known as 8602 [l I]) was used for most of this work. It belongs to Habs
Pseudomonas Lipopolysaccharide
214
serogroup 3 and carries a lysogenic bacteriophage PS1 (P. H. Clarke, personal communication). A spontaneous mutant resistant to this bacteriophage was isolated and designated PAClR. Its serotype and all other properties tested, except that of sensitivity to PS1, appeared to be unchanged from the parent strain PAC1. Strains P. aeruginosu 2A and 9 were the Habs type strains [9]. Colindale 9 was isolated from clinical material in the Central Public Health Laboratories, Colindale, London. Colindale 9PA was a spontaneous mutant selected by its resistance to bacteriophage 7 [12]. The bacteria were grown at 37 "C from a 1 % inoculum in 1-1 batches in 5-1 flasks shaken at 37°C or in a 1000-1 fermenter with an aeration of 300 l/min. The medium used was either nutrient broth (Oxoid no. 2) or minimal medium [13] supplemented with sodium succinate (0.5 %). When they reached the stationary phase of growth the bacteria were harvested by centrifugation at 15000 x g for 30 min, washed twice with distilled water and their walls prepared after disruption in a French pressure cell as previously described [4].
Isolation and Degradation of' Lipopolysaccharide Lipopolysaccharide was normally isolated from defatted walls by extraction with 45 "/, aqueous phenol [l]. The aqueous phases were centrifuged to remove contaminating peptidoglycan, dialysed and freezedried as described by Key et al. [14]; yield approx. 100 mg/g walls. Where specified the petroleum ether/ chloroform/phenol method [15] was also used. Lipid A was removed from the isolated lipopolysaccharide (25 mg) by hydrolysis with acetic acid [2]. The lipid was extracted with chloroform and the watersoluble products (partially degraded polysaccharides) freeze-dried. They were separated by elution from a column (3 x 55 cm) of Sephadex G-75 with pyridinel acetate buffcr (0.05 M, pH 5.4) [6]. Fractions (4 ml) were analysed for neutral sugar content by the phenol/ sulphuric acid method [ 171. For alkaline de-0-acylation lipopolysaccharide (50 mg) was suspended in 50 ml99 aqueous ethanol (0.1 N in NaOH) and incubated for 45 min at 37 "C with occasional shaking. The resulting product was neutralised with hydrochloric acid (2 N), evaporated to dryness at 4'C by rotary evaporation and the residue dissolved in about 3 ml of pyridine/acetate buffer (0.05 M, pH 5.4). The solution was centrifuged (I5000 x g , 5 min) to remove any insoluble material and the supernatant fluid filtered through sintered glass (no. 1 porosity). It was applied to a column (3 x 55 cm) of Sephadex G-200, eluted with pyridine/ acetate buffer, and fractions analysed with phenol/ sulphuric acid as before.
Anulytical Methods Methods used for the determination of total phosphorus and glucose and for the detection and estimation of amino compounds by automatic analysis were all as described previously [4]. No correction was made for the destruction or slow release of components during hydrolysis. The compound previously designated U4 [4] was identified by comparison with an authentic sample as fucosamine. The amounts of the unknown amino compounds were calculated assuming a colour yield of 1 and a molecular weight equivalent to that of glucosamine. 2-Keto-3-deoxyoctonic acid, rhamnose, fatty acids and heptose were all determined as described by Fensom and Gray [18]. Amounts of heptose were calculated as L-glycero-D-mannoheptose using the standard values obtained by Osborn [19]. RESULTS The lipopolysaccharide analysed previously [2] had been isolated from whole organisms of P. aeruginosa PACl grown in 1-1 batches in minimal medium with citrate as carbon source. Lipopolysaccharide isolated from walls of this strain grown in 1-1 batches in nutrient broth was very similar both in yield and composition (Table 1, batch 1). The main sugar components were glucose, rhamnose, heptose and 2-keto3-deoxy-octonic acid. The amino compounds present, glucosamine, glucosamine phosphate, galactosamine, fucosamine and two unidentified amino compounds designated U2 and U3, had all previously been detected in lipopolysaccharide isolated from PACl and another Habs serotype 3 strain [4]. In order to obtain enough material to analyse in detail, we grew larger batches in a 1000-1 fermenter and, to prevent induction of the lysogenic bacteriophage PS1, used a spontaneous mutant resistant to it, PAClR. The lipopolysaccharide prepared from walls of this mutant grown in 1-1batches in nutrient broth and minimal medium with succinate as carbon source (Table 1, batches 2 and 3) had very similar chemical composition to that of PACl . Similarly lipopolysaccharide isolated from PACl R grown in 500 1 of nutrient broth in the fermenter had the same composition (Table 1, batch 4). However when a 500-1 batch was grown in minimal medium with succinate as carbon source the lipopolysaccharide isolated was quite different (batch 5) and will be described as abnormal lipopolysaccharide. It was deficient in heptose, glucose, alanine, galactosamine and glucosamine phosphate as compared with the other preparations analysed. It also contained particularly large amounts of a material previously detected by automatic amino compound analysis in hydrolysates of normal polysaccharide from PACl and another strain of Habs type 3 serotype [4]. This had a high
I. R. Chester and P. M. Meadow
215
Table 1. Elfect of growth conditions on the composition of lipopolysaccharide from P.aeruginosa PACl and PACIR Results are expressed as percentages of lipopolysaccharides ~
Component
Glucose Rhamnose Heptose Phosphorus Glucosamine phosphate A I an i n e Glucosamine Galactosamine U2" u3 a Fucosamine 2-Keto-3-deoxyoctonic acid "
Amount present in batch
1 (PACl in 1-1 nutrient)
2 (PAClR in 1-1 nutrient)
3 (PACIR in 1-1 succinate)
4 (PACIR in 500-1 nutrient)
5 (PAClR in 500.1 succinate)
10.2 9.6 4.2 4.6 1.6 1.1 6.8 1.5 1.8 0.1 trace 3.4
8.4 11.6 5.6 4.8 1.5 1.2 8.0 1.8 1.6 0.2 0.2 2.4
9.9 11.9 5.2 4.4 1.8 1.2 7.8 1.6 1.4 0.2 0.1 2.4
12.8 10.9 4.4 4.5 2.6 1.3 8.6 1.6 1.6 0.4 trace 2.9
0.3 15.9 0.4 1.o 0.4 0.4 13.2 0.5 2.8
0.6 trace 0.9
Unidentified amino compounds [4].
absorbance at 440 nm after ninhydrin treatment and was basic, being eluted just after galactosamine. It has not yet been identified. The low heptose content of the abnormal lipopolysaccharide suggested that if might have less than the normal amount of lipid A. When it was treated for 60 min with 1 yo acetic acid which precipitates lipid A from normal lipopolysaccharide of PACl R and PAC1, there was no precipitate, although prolonged hydrolysis caused slight cloudiness. Treatment with 0.1 N HC1 which splits lipid A from some pseudomonad lipopolysaccharides [4] also failed to release appreciable amounts of lipidA from the abnormal lipopolysaccharide. However by increasing the amount of abnormal lipopolysaccharide three-fold (100 mg) and hydrolysing with 1 % acetic acid at 100°C for 150 min, sufficient lipid A was released for analysis. The fatty acids present were those previously identified in lipid A from PACl [20] but the amount (6.8 %) was lower than in the normal lipopolysaccharide (20.9 "/,). One possible explanation of the unusual composition of the abnormal lipopolysaccharide would be that under certain conditions a lipopolysaccharide was produced which was not readily extracted by the hot phenol method used. The rough lipopolysaccharidcs of the Enterobacteriaceae require a petroleum ether/ chloroform/phenol mixture for extraction [151. However walls of P. aeruginosa from which either normal or abnormal lipopolysaccharide had been extracted by aqueous phenol contained no detectable heptose or rhamnose which might indicate unextracted lipopolysaccharide. Furthermore, extraction of the dried walls with petroleum ether/chloroform/phenol only 1 of the lipopolysaccharide obtained by aqueous
phenol, although lipopolysaccharide isolated using the aqueous phenol method was soluble in petroleum ether/chloroform/phenol. Neither heptose nor rhamnose were detected in the phenol phase or insoluble residue after extraction of walls with aqueous phenol. There was thus no evidence for additional heptosecontaining or rhamnose-containing polymers in the walls other than those extracted into the aqueous phenol. A number of other growth conditions have been studied but abnormal lipopolysaccharide has been isolated only from cultures grown in the 1000-I-fermenter with succinate or lactate as carbon source. It has not so far been possible to determine the factors controlling abnormal lipopolysaccharide production. Acetic Acid Degradation of PACIR Lipopolysaccharide
The differences between the normal and abnormal PACl R lipopolysaccharides were even more striking after hydrolysis with 1 acetic acid and fractionation of the partially degraded polysaccharides on Sephadex G-75. The normal lipopolysaccharide like that from the Habs type 3 strain studied previously [4] was degraded into two major high-molecular-weight polysaccharide fractions distinct from the low-molecularweight fractions (Fig. 1A). 3-Keto-3-deoxyoctonic acid and phosphate were released as low-molecularweight solutes eluted after fraction L. Although acetic acid treatment of the abnormal lipopolysaccharidc released little lipid A, polysaccharides were released and could be fractionated into high and low-molccularweight components (Fig.1B). Most of the carbohydrate-containing material was eluted as a single
Pseudomanas Lipopolysaccharide
216
l.*IB
1.2 -
aH
A 1
.o -
1
:0.8
L
0.8 -
0.6 -
L
5
a
5
9 0.4 0.2
-
-
9g 0.6
8 * +
.o
0
2
0
0.4-
0.2
160
80
240
-
0' 0
320
i I
80
Eluate (rnl)
I
1
I
160 240 Eluate (ml)
320
Fig. 1, Fractionation on Seplzadex G-75 of pol.vsu(;charide releused by aci>tic trcatment of lipopolysacchuride Jrom P. aeruginosa PAC1 R . Bacteria were grown in 500-1 batches in (A) nutrient broth (normal) and (B) minimal medium with succinate as carbon source (abnormal). Lipopolysaccharide was extracted, purified and hydrolysed with 1% acetic acid as described in Methods. The partially degraded polysaccharidc was eluted from a column (3 x 55 cm) of Sephadex G-75 with pyridine acetic acid buffer pH 5.4.Fractions (4 ml) were analysed for total carbohydrate by the phenol/sulphuric acid method [17]
Table 2. Composition 0f;fractions obtained qfiw acetic acid hydrolysis of normal and uhnormal lipopolysaccharide of P. aeruginosa PAC I R The fractions analysed are shown in Fig. 1 Component
Amount present in high-molecular-weight fractions normal
Glucose Rhamnose Heptosc Phosphorus Glucosaminc phosphate Alanine Glucosamine Galactosamine u2 u3 Fucosamine
low-molecular-weight fractions abnormal
normal
abnormal
H1
H2
aH
L
aL
0.8 18.8 0 0.4 0 0.2 11.1 trace 3.4 0.6 0.6
9.7 28.9 0 0.5 0 0.9 2.1 0.7 0.5 trace trace
1.2 22.2 0 0.2
33.5 6.2 8.4 3.0 0.2 1.6 0.4 2.5 0.2 0.9 trace
16.8 7.2 trace 1.8 0 0.9 trace 1.2 trace 0 0
high-molecular-weight peak (aH) with smaller amounts of lower-molecular-weight material (aL). The major high-molecular-weight fractions (H1 and aH) from the normal and abnormal lipopolysaccharides contained most of the rhamnose glucosamine and fucosamine and unknown amino compounds U2 and U3 of the original lipopolysaccharides (Table 2). The low-molecular-weight fractions contained most of the glucose, heptose, alanine and galactosamine. That from the normal lipopoly-
0
0.3 16.7 trace 4.9 trace 0.8
saccharide (L) contained appreciable amounts of heptose, glucosamine and glucosamine phosphate, but these compounds were present in very much smaller amounts in fraction aL from the abnormal lipopolysaccharide (Table 2). The identifiable components in Table 2 account for only 28-56';/, of the polysaccharide fractions. The unidentified 440-nm absorbing materials present in particularly large amounts in the abnormal lipopolysaccharide were the only other compounds found.
I. R. Chester and P. M. Meadow
211
0.8 r A aN3
0.6
-
0.4
-
a
E 0.6
8-
.J c
m
c
m
a,
e
e 5:
9
0.4
c
0.2
8
-
n -0
I
80
I
I
I
160 240 Eluate (ml)
320
0.2
0
0
80
160 240 Eluate (ml)
320
Fig. 2. Fractionation on Sephadex G-200 of polysaccharide obtained by de-0-acylation bitith ethanolic alkali of lipopolysaccharide ,from P. aeruginosa P A C I R . Bacteria were grown and lipopolysaccharide extracted as described under Fig. 1. Lipopolysaccharide was treated with ethanolic alkali and fractionated on a column of Sephadex (3-200 as described in Methods. Fractions were analyscd for total carbohydrate
Alkali Degradation of PAC1 Lipopolysaccharide If the normal lipopolysaccharide consisted of more than one type of molecule, then the polysaccharide fractions H1 and H2 obtained by acetic acid hydrolysis might have been derived from different lipopolysaccharides one of which, that yielding H2, was not made under the conditions producing abnormal lipopolysaccharide. The isolated lipopolysaccharides were therefore treated with ethanolic alkali before fractionation. This treatment removes 0-acylated fatty acids from the lipid A [21] thus reducing non-polar interactions between lipopolysaccharide components and facilitating their separation. It was hoped that the mild conditions would not degrade the lipopolysaccharides further. Separation of the products of alkaline degradation on Sephadex G-200 gave several fractions from both the normal and abnormal lipopolysaccharides (Fig. 2 A and B). The lowest-molecular-weight fraction of the normal lipopolysaccharide (N4) which accounted for about half the material applied, was completely absent from the abnormal lipopolysaccharide. This was perhaps to be expected since the more drastic acetic acid treatment had also released very little low-molecular-weight material. Analysis of the fractions obtained from the normal lipopolysaccharide showed that they all contained those components concentrated in the low-molecularweight fractions produced by acetic acid degradation ; that is glucose, heptose, alanine, galactosamine and phosphate (Table 3). This suggested that the normal lipopolysaccharide might consist of 3 or 4 types of macromolecules each of which contained the same core fraction with additional high-molecular-weight fractions attached. If so then N4 might be most closely related LO the basic molecule and might correspond to the rough core of the Enterobactcriaceae. Hydrolysis of N4 with acetic acid followed by elution from Sephadex G-75 yielded a polysaccharide whose elution properties and composition were indistin-
Table 3 . Compositions of fractions obtained by elution fiom Sephadex G-200 of material released by ethanolic alkali de-0-acylution of normal 1ipopol.vsaccharide of PAClR Fractions are those shown in Fig. 2 A Component
Amount in fraction N1
N2
N3
N4
0.4 20.5 1.1 1.3 0.7 0.4 12.9 0.6 3.2 0.6 trace 3.3
1.9 19.9
16.9 5.1 5.3 5.7 2.9 2.4 6.2 3.6 0.2 0.1 trace
o/ /"
Glucose Rhamnose Heptose Phosphorus Glucosamine phosphate Alanine Glucosamine Galactosamine u2 u3 Fucosamine 2-Keto-3-deoxyoctonate
6.6 11.4 2.3 2.4 1.9 1.4 11.2 1.9 1.9 0.3 trace 1.7
2.1 1.8
1.3 0.8 4.9
1.4 0.8 0.2 trace 1.8
4.3
guishable from fraction L prepared by acetic acid hydrolysis of the intact lipopolysaccharide. Both N4 and L contained relatively large amounts of glucose suggesting that they might be contaminated with a glucose polysaccharide. However, although fraction L could be further separated by Biogel P 4 into two fractions, both contained glucose linked to the other sugar components (Chester and Meadow, unpublished). It seems unlikely therefore that there is much chemical heterogeneity in the common core polysaccharide. Although fractions N1, N2 and N3 all appeared to contain the same core polysaccharide found in N4 and L, the amounts of polysaccharide common to each fraction must vary since the proportions of the core components varied. These fractions also contained those substances previously found in thc acetic acid degradation products H1 and H2,
278
Pseudomonas Lipopolysaccharide
Eluate (ml)
Eluate (ml)
Fig. 3. Fractionalion q/ purtiully degruded lipop(~ly.~ac~l~uride Jrom P. aeruyinosa 2 A . (A) Acid-hydrolysed products separated on Sephadex G-75. (B) Hydrolysed by ethanolic alkali and separated on Sephadex G-200. Fractions were analysed for total carbohydrate
that is rhamnose, glucosamine, U2, U 3 and fucosamine, U2, U3 and fucosamine. Hydrolysis of N1, N2 and N 3 with 1 "/, acetic acid and separation of the polysaccharides released on Sephadex G-75 confirmed their relationship to H1, H2 and L. N1 and N2 both yielded a major component with identical elution properties to H1 together with smaller amounts of components behaving as H2 and L. N 3 gave mainly material resembling H2 with smaller amounts of L but the amounts of material were too small to analyse. The fractions isolated after de-0-acylation of the abnormal lipopolysaccharide (aN1, aN2 and aN3) (Fig. 2 B) contained the high levels of rhamnose, glucosainine and unknown amino compound U2 characteristic of the acetic acid fraction a H from the same material. The compositions of all three fractions were similar to N 1 and N2 obtained from the normal lipopolysaccharide (Table 4). The presence of appreciable amounts of glucose may suggest a closer resemblance t o N1 than to N2. However the amounts of alanine and glucosamine in aN1, aN2 and aN3 were much less than in N 1 and N 2 and heptose was not detected. This suggests that the amount of core polysaccharide was very much reduced in each of the fractions obtained from the abnormal lipopolysaccharide, as had been indicated by the acetic acid degradation products. Lipopolysaccharide,from Other Strains of P . aeruginosa
The lipopolysaccharides of the Habs type 3 strains to which PAS1 belongs appear to be the most complex of all the strains examined as judged by their hydrolysis products [4]. It was therefore not surprising that alkaline degradation confirmed the complexity of
Table 4. Composition q[frartions obtained by elution from st pi rude.^ G-200 of material released by ethanolic alkali de-0-acylation qf ' abnormal lipopolysaccharide of PAClR Fractions are those shown in Fig.2B Cornponcnt
Amount in fraction aN1 I,
"Y'
Glucose Rhamnose Heptose Alanine Glucosamine Galactosamine u2 u3 Fucosdmine
aN2
aN3
1.3 16.1 0 kdCe 13.1 trace 2.1 2.5 trace
2.6 14.6 0 0.3 12.0 trace 1.8 0.3 trace
,
3.3 8.1 0 0.4 5.1 0 1.o 0 kdCe
PAC1 lipopolysaccharide. It was interesting to see whether there was any evidence for heterogeneity in strains whose lipopolysaccharides yielded fewer products after acid hydrolysis. One of the simplest elution profiles was obtained after acid hydrolysis of lipopolysaccharide from type 2A. Separation of the products on Sephadex G-75 yielded a single low-molecularweight peak (2AL) containing all the neutral sugars of the intact lipopolysaccharide (Fig. 3A). The only material detected in the high-molecular-weight fractions (2AH) were amino compounds. De-0-acylation of the 2A lipopolysaccharide followed by separation of the products on Sephadex G-200 also produced a much simpler pattern than had been found with PAC1 lipopolysaccharide (Fig. 3B). Most of the neutral
I. R. Chester and P. M. Meadow
219 1.2
0 ColSPAN3 1
.o
A
C
9N3
COl9 N2
a
0.8
-3
c
a,
c" m
0.6
5
f \
COlSPANl
3
c
8
p
0.4
0.4
0
P 0.2
80
160
240
320
0
0.2
60
Eluate (ml)
n 160 Eluate ( m l )
240
320
0
80
160
240,
320
Eluate ( m i )
Fig. 4. Fruc,rionation on Sephudu G-200 of partially degraded lipopolysucchuride ,from P. aeruginosa Colindule 9 ( A ) , Colindale YPA ( B ) unii Hubs type Y ( C ) . Lipopolysaccharide was treated with ethanolic alkali and separated on Sephadex G-200 as described in Methods. Fractions were analyscd for total carbohydrate
Table 5 . Di.stributionof amino compounds between fractions separated after arid and alkaline degradation of type 2A lipopolysaccharide The fractions andlysed are those shown in Fig.3. Results are expressed as percentages of the total detected Component
Amount in fraction high-molecularweight
low-molecularweight
2AH
2AL
2AN2
0 YY.8 0
91.1 87.9 86.9
100 5.6 53.4
XY.0
2AN1
0
Glucosaminc phosphate 0 0.2 Alanine Ghcosdmine 0 Galactosaminc < 0.1 94.4 u3 Fucosamine 46.6
8.3 12.1 13.1 11.0 100 47.8
0 52.2
sugars were eluted as a single peak (2AN2) with elution properties and composition very similar to that obtained from PACl (N4, Fig. 1A). The highmolecular-weight fraction (2AN 1) contained small but significant amounts of glucose, heptose, alanine and galactosamine thought to be characteristic of the core polysaccharide. The distribution of the amino compounds between high and low-molecular-weight fractions was very similar following either acid or alkaline degradation except for the loss of glucosamine from the acid fractions. Presumably glucosamine forms part of the lipid A removed by acid hydrolysis. The alanine and galactosamine were concentrated in the low-molecular-weight fractions, U 3 was mainly in the high-molecular-weight fractions and fucosamine
was equally distributed between the two (Table 5). These results suggest that in type 2A as in PAC1 lipopolysaccharide there is more than one structure. The one with lower molecular weight gives rise to 2AL or 2AN2 after acid or alkali treatment. The other contains mainly amino compounds with a smaller amount of the polysaccharide similar if not identical to that of lower molecular weight. Lipopolysaccharides from other strains of P. aeruginosa were also de-0-acylated by treatment with ethanolic alkali and their degradation products separated by elution from Sephadex G-200 (Fig. 4). Each yielded a major component behaving as N4 from PACl together with smaller amounts of highermolecular-weight materials. Those which had yielded complex elution profiles after acid hydrolysis [4] gave several components after alkaline hydrolysis while those with simpler acid hydrolysis products yielded few. Lipopolysaccharide from Habs type 9 which had proved resistant to acetic acid hydrolysis was readily split by ethanolic alkali into at least three components (Fig. 4C). The major product ofthe de-0-acylated lipopolysaccharide from each of the strains was very similar both in elution properties and chemical composition (Table 6). The only differences detected were in the amounts and character of the minor amino compounds. These amino compounds were the same as those previously detected in acid hydrolysed lipopolysaccharide and thought to be characteristic of the strain and possibly the serotype [4]. The other de-0acylation products all contained the common core components as well as the amino compounds detected in the high-molecular-weight fractions obtained by acid hydrolysis of the same lipopolysaccharide.
280
Pseudomonas Lipopolysaccharide
Table 6. Composition of major fractions separated on Sephadex G-200from de-O-acylated lipopolysaccharides of different strains ofP. aeruginosa The fractions analysed are shown in Fig. 2A, 3 A and 4. The amino compounds U1,2,3,5,6,7 are those previously described in these strains [4] Component
Amount in strain (serotype, fraction) .
Glucose Khamnose Heptose Glucosamine phosphate Alanine Glucosamine Galactosaminc
u1 u2 u3 Fucosamine
~
~~~
PAC 1R (3, N4)
2A (2A, 2AN2)
Colindalc 9 (6, Co19N2)
C,olindale 9PA (not typable, Col9PAN3)
9
16.9 5.1 5.3 2.9 2.4 6.2 3.6 0 0.2
14.6 3.0 5.5 2.7 2.4 4.9 3.4 0 0 trace 0.4 0
15.6 6.9 4.4 1.7 2.0 4.1 2.9 0.3 1.6 1.9 3.4 2.7
19.5 4.6 6.1 2.3 2.2 4.6 3.6 tracc 0.6 0.3 1.8 0.9
15.5 5.3 4.7 2.4 1.9 3.9 2.5
0.1
trace 0
DISCUSSION The phenol-extracted lipopolysaccharides of several strains of P. aeruginosa have now been analysed in a number of laboratories and their overall composition found to be very similar [2,4-6,8,9,22]. They can be partially degraded by acetic acid and separated into fractions of differing molecular weight. The lowermolecular-weight fractions appear similar if not identical in all strains and may represent a common core polysaccharide similar to that found in the Enterobacteriaceae. The high-molecular-weight fractions varied between strains and some at least of this variation may depend on their O-serotypes. In strain PACl and its bacteriophage-resistant mutant PAClR the high-molecular-weight fractions separated after acetic acid hydrolysis contained both neutral sugars (mainly rhamnose and glucose) and most of the glucosamine and unidentified amino compound U2 of the complete lipopolysaccharide. Wilkinson and Galbraith [8] have found an amino compound X with similar properties to U2 in high-molecular-weight fractions of lipopolysaccharides from other strains of P. aeruginosa including one of Habs serotype 3. They suggest it may be an aminohexuronic acid. Most of these minor amino components of the lipopolysaccharides seem to be concentrated in the highermolecular-weight fractions obtained after both acid and alkaline degradation of the strains studied here. Aminosugar-rich fractions have also been obtained by deoxycholate treatment of P. aeruginosa P14 (serotype 1) [6]. Again these fractions were of higher molecular weight than the fractions rich in neutral sugars. The role of the high-molecular-weight aminosugarrich fractions of the lipopolysaccharides in the pheno-
(9, 9N3)
0 0
3.3 2.9 0
type of thePseudomonads is difficult to assess. Mutants defective in these fractions appear to have lost their serological specificity [7] (and Koval and Meadow, unpublished) and isolated high-molecular-weight fractions react with homologous antisera [4,8]. However there is not yet enough evidence to link specific amino compounds or groups of compounds conclusively with Habs serotype. On the other hand Suzuki has suggested that the presence of quinovosamine in lipopolysaccharides of P. aeruginosa may confer sensitivity to pyocins A and S. A pyocin-resistant mutant lacks quinovosamine which occurs solely in the highermolecular-weight fractions of the lipopolysaccharide [22]. In the few available mutants of PACl which are defective in high-molecular-weight fractions of the lipopolysaccharide we have so far been unable to find any common phenotype. The overproduction of high-molecular-weight fractions of the lipopolysaccharide of PAClR under certain growth conditions is difficult to interpret particularly as we have been unable to define the cause. The cultures producing abnormal lipopolysaccharide had the same growth characteristics and produced the same final yield as those producing the normal lipopolysaccharide. The bacteria had normal morphology and were indistinguishable from the wild type by any of the biochemical or genetic tests used. The production of abnormal lipopolysaccharide appears to be phenotypic rather than genotypic. It may simply be an extreme example of the heterogeneity normally apparent in pseudomonas lipopolysaccharide and result from changes in the control of lipopolysaccharide synthesis. However although the highmolecular-weight fractions obtained from alkaline degradation of the abnormal lipopolysaccharide were
1. R. Chester and P. M. Meadow
similar to those from the normal there were some differences. The content of core pol ysaccharide was much less than in the normal fractions and lacked the heptose found in the latter. Changes in heptose content of dried walls of Aerohacter aerogenes were shown by Ellwood and Tempest to be related to growth conditions [23]. The lowest heptose contents were obtained in glycerol-limited cultures using low dilution rates. In preliminary experiments using similar conditions we have so far been unable to produce similar results in P.aeruginosu PACl and we do not know what causes production of abnormal lipopolysaccharide with low heptose content under some circumstances in a 1000-1fermenter. In the Enterobacteriaceae heptose is part of the core to which the lipid A and common and antigen-specific polysaccharides are attached [3] and a similar structure has been suggested for P. aerughosa 1999 [9]. It is difficult to see how the abnormal lipopolysaccharide of PACl could be reconciled with this type of structure and more detailed chemical analysis will be required. One of the unusual features of the pseudomonad lipopolysaccharide is the relatively low (16 - 24 o/,) carbohydrate content [4,8]. Even in the isolated polysaccharide fractions we found that only 24- 45 % of the dry weight could be accounted for as carbohydrate. This is near the range (32- 50 %) found by Wilkinson and Galbraith in similar fractions from other strains of pseudomonas [8]. Our analyses did not include ethanolamine, known to form part of the intact lipopolysaccharide [ 5 ] , nor the 440 nm absorbing materials detected by automatic amino compound analysis. Furthermore no correction was made for degradation during hydrolysis. Some of the phosphate is acidlabile [ 5 ] and if U2 is an aminohexuronic acid its content could have been seriously underestimated. Nevertheless a substantial part of the lipopolysaccharide and of its polysaccharide fractions has not been identified particularly in the abnormal lipopolysaccharide from PAC1. We have no evidence as to what the components might be. Both acid and alkaline degradation products of the lipopolysaccharides studied support the view that all strains of P.aeruginosa contain a common core polysaccharide. Specific amino compounds are attached to this in amounts varying from one strain to another and the proportion of the two parts may also vary with physiological conditions. In addition most strains appear to contain other lipopolysaccharides consisting of those components previously detected in the high-molecular-weight acid-degradation products attached to smaller lengths of core polysaccharide. In some strains, particularly PACl and Habs type 9 there was evidence for more than one type of highmolecular-weight fraction possibly corresponding to more than one type of side chain.
28 1
On the lirnitcd evidence available it is difficult to construct a picture of the lipopolysaccharide of P . aeruginosa. The similar fatty acid, phosphate and 2-keto-3-deoxyoctonate contents of the lipopolysaccharides isolated from several strains of P . cieruginosu [4,8] suggest a common lipid A component and the yield of core (L) fractions is remarkably constant [4]. The major alkaline degradation products for different serotypes were very similar except for the specific amino compounds. It is possible that the structure consists of lipid A linked through 2-keto3-deoxyoctonate to the core polysaccharide. To some but not all of these molecules additional side chains could be attached and there may be variation in the numbers of core repeating units. In PACl the normal lipopolysaccharide may consist of at least three types of molecule, one of which contains lipid A linked to the core polysaccharide, a second has the H1 polysaccharide added to this basic molecule while the third has the H2 polysaccharide linked to the core and thence to lipid A. The only lipopolysaccharide-defective mutants we have so far been able to isolate from PACl appear to have lost both the H1 and H2 polysaccharides in a single step (S. Koval and P. M. Meadow, unpublished results). This could mean that H1 and H2 are part of the same lipopolysaccharide molecule, but could also result from a defect in the core. Until more is known of their structure it is not possible to distinguish between the alternatives. We are grateful to the Medical Research Council for a project grant which allowed us to undertake this work. We also thank Mr P. D. Laverack who carried out the automatic amino compound analyses and Mr C. J. Hubball and Mr P. Wells for excellent technical assislance.
REFERENCES 1. Westphal, 0. & Jann, 0. (1965) Meth0d.s Carhohyd Chenz. 5, 83-91. 2. Fensom, A. H. & Meadow, P. M. (1970) FEBS Lert. 9,81- 84. 3. Luderitz, 0..Westphal, 0..Staub, A. M. & Nikaido, H . (1971) Microbial Toxins IV, p. 145, Academic Press, London. 4. Chester, I. R., Meadow, P. M. & Pitt, T. L. (1973) J . Gen. Microhiol. 78, 305-318. 5. Drewry, D. T., Lomax, J. A,, Gray, G. W. & Wilkinson, S.G. (1973) Biochem. J. 133,563 - 512. 6. Ikeda, K. & Egani, F. (1973) J . Gen. Appl. Microbial. 19. 115- 128. 7. Meadow, P. M. (1975) Genetics and Biochemistry of’ Pseudomonas, p. 67, John Wiley & Sons, Chichester. 8. Wilkinson, S. G. & Galbraith, L. (1975) Eur. J . Biochem. 52, 331 -343. 9. Drewry, D. T., Symes, K. C., Gray, G. W. & Wilkinson, S.G . (1975) Biochem. J. 149, 93-106. 10. Habs, I. (1957) Zentralbl. Bakteriol. Parasitenkd. Injektionskr. Hyg. Abt. I Orig. 144, 218-228. 11. Brown, P. R. & Clarke, P. H. (1972) J . Gen. Microhiol. 70, 287-299. 12. Martin, D. R. (1971) Ph.D. Thesis, University of London.
282 13. Clarkson, C. E. & Meadow, P. M. (1971) J. Gen. Microhiol. 66,161 - 169. 14. Key, B. A,, Gray, G. W. & Wilkinson, S. G. (1970) Biochem. J . 120, 55Y - 566. 15. Galanos, C., Luderitz, 0. & Westphal, 0. (1969) Eur. J . Biochem. 9,245- 249. 16. Schmidt, G., Jann, B. & Jann, K. (1969) Eur. J . Biochem. 10, 501 -510. 17. Dubois, M., Gilles, K. A,, Hamilton, J. K., Rebers, P. A. & Smith, F. (1956) Anal. Chem. 28,350-356.
I. R. Chester and P. M. Meadow: Pseudomonas Lipopolysaccharide 18. Fensom,A. H. & Gray, G . W . (1969) Biochern. J. 114, 185196. 19. Osborn, M. J. (1963) Proc. Nut1 Acad. Sci. U.S.A. 50, 499506. 20. Hancock, I. C., Humphreys, G. 0. & Meadow, P. M. (1970) Biochim. Biophys. Acla, 202, 389-391. 21. Kasai, N. (1966) Ann. N.Y. Acad. Sci. 133, 486-507. 22. Suzuki, N. (1974) FEBS Lett. 48, 301 -305. 23. Ellwood, D. C. & Tempest, D. W. (1972) Adv. Microh. Physiol. 7 , 83- 117.
I. R. Chester, Fermentation Development Laboratory, Beecham Pharmaceuticals, Clarendon Road, Worthing, Sussex, Great Britain P. M.Meadow, Department of Biochemistry, University College London, Gower Street, London WClE 6BT, Great Britain
