Extracellular Microbial Polysaccharides

4 Microbial Exopolysaccharide Synthesis

Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch004

I. W. SUTHERLAND Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JG, Scotland

The fate of a carbohydrate (or other) substrate supplied to an exopolysaccharide-producing microbial cell depends on the microbial species chosen. As most results have been obtained from bacterial species, this review will be concerned essentially with the synthesis of exopolysaccharides by bacteria. In some bacteria, given the correct substrate, exopolysaccharide may be formed without penetration of the cell membrane by the substrate. This is seen in dextran and levan-forming cells supplied with sucrose or several of its analogues. Examples are to be found in Leuconostoc mesenterioides, Streptococcus or Bacillus species. Although this process has been studied by various workers, (1,2) the polysaccharides formed are more limited in their applications and current interest is centred rather on species which form their polymer intracellularly then excrete it into the medium. The aim is therefore to consider a series of processes by which substrates enter the microbial cells, are modified by a series of enzymic processes and finally are excreted in polymeric form from the microbial surface. Much of the information about these reactions has been gained from strains producing polymers which have little or no commercial value, but it is nevertheless possible to extrapolate many of the results and thereby obtain a reasonable hypothesis for the mode of synthesis of a polymer of given structure and to propose mechanisms for the regulation of its biosynthesis. Substrate Uptake The s u b s t r a t e may enter the c e l l by one of three mechanisms - f a c i l i t a t e d d i f f u s i o n , a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n . The l a t t e r two processes, both of which a r e endergonic, are of p a r t i c u l a r i n t e r e s t i n the present context. In a c t i v e t r a n s p o r t , the substrate enters the c e l l u n a l t e r e d , but the group t r a n s l o c a t i o n process i n v o l v e s the phosphorylation o f the s u b s t r a t e , the o v e r a l l process being represented by: X

+

PEP

• X-P

+

pyruvate

40

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

41

The i n i t i a l f a t e of the substrate i s summarised i n F i g . l . In E s c h e r i c h i a c o l i , the r a t e at which the b a c t e r i a grow on v a r i o u s substrates i s dependent on substrate uptake, i r r e s p e c t i v e of whether a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n systems are involved ( 3 ) . Thus substrate uptake i s one of the f i r s t l i m i t a t i o n s on exopolysaccharide production. As y e t , no attempts to increase c e l l growth and hence exopolysaccharide production by d u p l i c a t i o n of the genes concerned with a c t i v e t r a n s p o r t or with group t r a n s l o c a t i o n appears to have been made. In many b a c t e r i a , t h i s might not even be necessary, as s e v e r a l uptake mechanisms may e x i s t f o r each substrate i . e . Although a s p e c i f i c substrate may be transported by d i f f e r e n t mechanisms i n d i f f e r e n t microorganisms b a c t e r i a such as E* c o l i possess various mechanisms f o r uptake of a s i n g l e substrate such as g a l a c t o s e . D i f f e r e n c e s can c e r t a i n l y be expected between Gram p o s i t i v e and Gram negative b a c t e r i a or between pseudomonads and e n t e r i c s p e c i e s . The group t r a n s l o c a t i o n mechanisms i n v o l v i n g phosphorylation from PEP have been studied by Roseman and h i s colleagues (4) but i t i s not c l e a r whether the u t i l i z a t i o n of r e l a t i v e l y l a r g e amounts of PEP f o r substrate uptake lead to a r e d u c t i o n i n the amount of PEP a v a i l a b l e f o r other purposes. I f t h i s does r e s u l t under c o n d i t i o n s i n which growth i s l i m i t e d by substrate uptake and where high growth r a t e s are used, the r e s u l t might be a r e d u c t i o n i n the degree of p y r u v y l a t i o n observed i n the polymer excreted. Intermediary Metabolism and D i r e c t i o n to Polymer Synthesis F o l l o w i n g the entry of the substrate i n t o the c e l l and i t s phosphorylation by e i t h e r the group t r a n s l o c a t i o n mechanism or by a hexokinase u t i l i z i n g ATP, the substrate can be committed to e i t h e r anabolic processes or to m i c r o b i a l catabolism ( F i g . 2). I f i t s u f f e r s the l a t t e r f a t e , i t i s i n e f f e c t wasted as f a r as polymer production i s concerned, although i f i t enters the TCA c y c l e i t may be converted to pyruvate or to acetate and thus incorporated at a l a t e r stage i n t o polymer. The c o n t r o l of c a t a b o l i c processes w i l l not be considered here. The a n a b o l i c f a t e of the substrate can s t i l l take one of s e v e r a l l i n e s at t h i s stage. I f the m i c r o b i a l species under c o n s i d e r a t i o n i s a Gram negative species, forming exopolysaccharide, l i p o p o l y s a c c h a r i d e and glycogen, the carbohydrate may be converted to any one of these. In the p r o l i f e r a t i n g bacterium, glycogen i s r a r e l y synthesized, but i t s production i s a l s o d i f f e r e n t i a t e d from w a l l polymer or e x t r a c e l l u l a r polymer synthesis through the lack of involvement of i s o p r e n o i d l i p i d s . The c o n t r o l of glycogen synt h e s i s i s exerted through a l l o s t e r i c r e g u l a t i o n of ADP-glucose synthesis (5), the f i r s t enzymic step i n the pathway, which i s unique to glycogen synthesis ( F i g . 3). I t may thus be worth c o n s i d e r i n g the i s o l a t i o n of ADP-glucose pyrophosphorylase mutants i f the b a c t e r i a l s t r a i n i n which we are i n t e r e s t e d produces large


42

EXTRACELLULAR

SUBSTRATE

MICROBIAL

SUBSTRATE

POLYSACCHARIDES

SUBSTRATE

EXTRACELLULAR ENZYMES f POLYMER f

>

HISTIDINYL+ PEP +

PERMEASE


(DEXTRANS, L E V A N S , ETC.)

PROTEIN MEMBRANE

ENZYME M SUBSTRATE

SUBSTRATE - P H O S P H A T E

j KINASE + ATP SUBSTRATE -

+ PYRUVATE

PHOSPHATE + ADP Figure 1.

HEXOSE-

Initial pathways for extracellular substrates

•HEXOSE

6 Ρ—-HEXOSE

1 P- -CATABOLISM ENERGY

ANABOLISM POLYMERS Figure 2.

GLUCOSE

Fate of hexose substrate

G L C - 6 P — - G L C - 1 P UDP-GLUCOSE

PYROPHOSPHORYLASE

UDP-GLUCOSE

ADP-GLUCOSE PYROPHOSPHORYLASE

ADP-GLC

(GLC1^4GLC) GLYCOGEN EXOPOLYSACCHARIDE Figure 3.

LPS Anabolic fate of glucose


4.

SUTHERLAND

Microbial

Exopolysacchande

Synthesis

43


amounts of glycogen and thus converts s u b s t r a t e to an unwanted product. T h i s would e l i m i n a t e the " d r a i n " of glucose-l-phosphate i n t o glycogen synthesis and away from the d e s i r e d product. Such mutants would be p a r t i c u l a r l y v a l u a b l e i f a two-stage p r o d u c t i o n process was envisaged i n which the second stage contained c e l l s i n an e s s e n t i a l l y n o n - p r o l i f e r a t i n g environment, i . e . c o n d i t i o n s under which l a r g e q u a n t i t i e s of glycogen are normally s y n t h e s i z e d . ( S i m i l a r arguments would apply i f the micro-organism produce p o l y hydroxybutyric a c i d or t r e h a l o s e r a t h e r than glycogen.) The next precursor through which c o n t r o l can be exerted i s the sugar n u c l e o t i d e such as UDP-glucose. UDP-glucose pyrophosphorylase i s a key enzyme producing i n many micro-organisms a precursor f o r both w a l l polymers and exopolysaccharide b i o synthesis. The l e v e l of UDP-glucose pyrophosphorylase s y n t h e s i s appears to be almost u n a l t e r e d i n mutants d e f e c t i v e i n these polymers and t h i s i s r e f l e c t e d at l e a s t i n the E n t e r o b a c t e r i a c e a e , i n the l e v e l of UDP-glucose found i n n u c l e o t i d e pools of s e v e r a l strains (6). The s t r i c t c o n t r o l exerted by such enzymes as UDPglucose pyrophosphorylase or TDP-glucose pyrophosphorylase (7) enables some micro-organisms to channel intermediates to one p o l y mer or another. Thus, TDP-glucose i s a precursor of TDP-rhamnose: f o r i n c o r p o r a t i o n i n t o one or more polymers. I n species poss e s s i n g both enzymes mutual cross i n h i b i t i o n was observed, UDPglucose i n h i b i t i n g TDP-glucose pyrophosphorylase and TDP-glucose i n h i b i t i n g UDP-glucose pyrophosphorylase (7). T h i s could perhaps be p r e d i c t e d , as l o s s of s y n t h e s i s of p o l y s a c c h a r i d e would lead to the accumulation of both g l u c o s e - c o n t a i n i n g sugar n u c l e o t i d e s . T h i s double c o n t r o l i s apparently r e s t r i c t e d to micro-organisms i n which polymers c o n t a i n i n g both sugars are found and i s absent from micro-organisms l a c k i n g rhamnose-containing p o l y s a c c h a r i d e s . S i m i l a r c o n t r o l mechanisms are found i n the formation of fucose as GDP-fucose from GDP-mannose. T h i s was s t u d i e d i n b a c t e r i a l species c o n t a i n i n g ( i ) D-mannose i n t h e i r p o l y s a c charides; ( i i ) c o n t a i n i n g L-fucose; and ( i i i ) c o n t a i n i n g both D-mannose and L-fucose ( 8 ) . In the f i r s t type, c o n t r o l of the r a t e of GDP-mannose s y n t h e s i s occurred through GDP-mannose pyrophosphorylase. In those b a c t e r i a i n which GDP-mannose i s s o l e l y a precursor i n fucose s y n t h e s i s , GDP-fucose c o n t r o l l e d both GDPmannose pyrophosphorylase and GDP-mannose hydrolyase through feedback i n h i b i t i o n . When both mannose and fucose are present i n polysaccharides produced by a s i n g l e bacterium, each sugar nucleot i d e c o n t r o l l e d i t s own s y n t h e s i s ( F i g . 4 ) . Xanthomonas campestris i s of p a r t i c u l a r i n t e r e s t because GDP-mannose and UDPglucose most probably both serve as precursors f o r l i p o p o l y s a c charide and exopolysaccharide. Further c o n t r o l o f the n u c l e o t i d e pool can occur through UDPsugar hydrolases (9,10), although, as these enzymes i n E. c o l i are


44

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES


p e r i p l a s m i c , they may not n e c e s s a r i l y have access to a l l the sugar n u c l e o t i d e formed by a c e l l but may be s p a t i a l l y separated from i t i n normal c e l l s . As s e v e r a l of the enzymes i n v o l v e d i n sugar n u c l e o t i d e synthesis are membrane-bound, i t i s by no means c l e a r whether t h e i r products occur f r e e l y w i t h i n the cytoplasm or whether they are produced i n c l o s e proximity to the enzymes which r e q u i r e them f o r polymer s y n t h e s i s . There i s a l s o the p o s s i b i l i t y of genetic r e g u l a t i o n of precursors s p e c i f i c to a p a r t i c u l a r polymer. The example of t h i s which has probably r e c e i v e d most study, through the work of Markovitz and h i s colleagues (11,12,13), i s c o l a n i c a c i d synthesis i n c e r t a i n b a c t e r i a of the Enterobacteriaceae. Knowledge of the s t r u c t u r e of c o l a n i c a c i d (14,15) ( F i g . 5) r e v e a l s two monosacc h a r i d e s , D-glucose and D-galactose, common to exopolysaccharide and to w a l l polymers and two others, L-fucose and D-glucuronic a c i d , unique to the polymer. C o n t r o l of the exopolysaccharide synthesis involved r e g u l a t o r genes; mutations i n these genes l e d to derepression and increased polysaccharide s y n t h e s i s . As a r e s u l t of the derepression, increased production of the three enzymes l e a d i n g to GDP-fucose synthesis and under the c o n t r o l of one r e g u l a t o r gene, was detected; increased formation of UDPglucose dehydrogenase ( r e s p o n s i b l e f o r conversion of UDP-glucose to UDP-glucuronic acid) occurred from mutation i n another r e g u l a t o r gene. As y e t , the concept of such r e g u l a t o r genes as those found i n c o l a n i c a c i d formation, dominant on episomes but r e c e s s i v e when located on the b a c t e r i a l chromosome, i s confined to a few s t r a i n s of E. c o l i , Salmonella e t c . One should not discount the p o s s i b i l i t y that polysaccharide production i n other genera and species i s under s i m i l a r genetic c o n t r o l , e s p e c i a l l y as so l i t t l e i s known about the genetic systems of most exopolysacchar ide-producing micro-organisms. Formation of Exopolysaccharide The c o n s t r u c t i o n of the r e p e a t i n g u n i t s of the polymer i s dependent on t r a n s f e r of the appropriate monosaccharides from sugar n u c l e o t i d e s to a c a r r i e r l i p i d i s o p r e n o i d a l c o h o l phosphate. The sequence of r e a c t i o n s has been w e l l c h a r a c t e r i z e d through i s o l a t i o n of the products at each t r a n s f e r step (16) and through i s o l a t i o n and i d e n t i f i c a t i o n of mutants (17) i n two Enterobacter aerogenes systems. The s e r i e s of r e a c t i o n s f o r the s t r a i n studied by Troy et ail. (16) was: UDP-Gal

+ P-lipid « = ±

+

UMP

Gal-P-P-lipid

+ GDP-Man

• Man-Gal-P-P-lipid

+

(GDP)

Man-Gal-P-P-lipid

+ UDP-GlcA

• GlcA-Man-Gal-P-P-lipid +

(UDP)

y Gal-Man-Gal-P-P-lipid

(UDP)

GlcA-Man-Gal-P-P-lipd + UDP-Gal

Gal-P-P-lipid

GlcA


+

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

f" — s


Man - I - Ρ — - — - G D P - M a n ii)

Man- l-P J-GDP-Man

iii)

Man- I - P-^GDP-Man

- POLYMER

(-Man-)

n

GDP - Fuc - * P O L Y M E R ( - F u c - ) ^

-i

GDP - F u c — P O L Y M E R S (-Man-)

1 = G D P - m a n n o s e pyrophosphorylase 2=

'

F

u

C

'

n n

GDP-mannose hydro-lyase

3= G D P - f u c o s e s y n t h e t a s e Figure 4. Control of mannose and fucose synthesis (after Kornfeld Gloser, 1966)

and

Ί

P Y R U V = ^ Gal 4 β GIcA 1 '3 Gal

3 \ β Glc 1

I

»· F u d

Figure 5

t

*4Fuc 1

Ac


n


46

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

In the other s t r a i n studied (17), the f i r s t r e a c t i o n a l s o involved t r a n s f e r of a hexose-l-phosphate. The methods employed i n these studies l i m i t e d the s i z e of fragment which could be i d e n t i f i e d as being attached to the l i p i d . The l a r g e s t o l i g o s a c c h a r i d e charac t e r i z e d was an octasaccharide equivalent to two r e p e a t i n g u n i t s (16). The exact mechanisms involved i n f u r t h e r chain e l o n g a t i o n and e x t r u s i o n of exopolysaccharides i s s t i l l unknown. Recently, two of the enzymes involved i n Ε. aerogenes have been shown to be extremely l i p o p h i l i c p r o t e i n s e x t r a c t a b l e from membrane prepara t i o n s with a c i d butanol. In t h i s they resemble the i s o p r e n o i d a l c o h o l phosphokinase p u r i f i e d e a r l i e r from Staphylococcus aureus (18) and a s i m i l a r but not i d e n t i c a l p r o t e i n prepared from E. aerogenes (19,20). The s i t e of s y n t h e s i s of l i p o p o l y s a c c h a r i d e , which shares the requirement f o r c a r r i e r l i p i d and a l s o f o r c e r t a i n of the sugar n u c l e o t i d e s , has been i d e n t i f i e d as the cytoplasmic membrane (21). P r e l i m i n a r y experiments i n our labo .ratory have shown that i t i s a l s o the s i t e of exopolysaccharide synthesis (Table 1). Attempts to p u r i f y the t r a n s f e r a s e enzymes by detergent s o l u b i l i z a t i o n were u n s u c c e s s f u l ; membrane p r o t e i n s were s o l u b i l i z e d but the procedure u s u a l l y l e d to p a r t i a l or complete i n a c t i v a t i o n . Although studies of t h i s k i n d have only been a p p l i e d to a l i m i t e d number of micro-organisms, the general mechanisms appear to be the same. In the synthesis of the phosphorylated mannan °f Hansenula capsulata, both mannose and phosphate were derived from GDP-mannose (22). Although i n t h i s p a r t i c u l a r study there was no attempt to demonstrate the involvement of l i p i d i n t e r mediates, they f u n c t i o n i n the formation of s i m i l a r polymers i n m i c r o b i a l w a l l s (23). As the enzyme preparations used i n these studies were crude membranes, nothing i s known about t h e i r r e g u l a t i o n , although i n a s e r i e s of non-polysaccharide-forming Ε. aerogenes mutants, the amount of t r a n s f e r a s e a c t i v i t y appeared to be lower than that found i n w i l d type b a c t e r i a (17). Isoprenoid L i p i d s i n Exopolysaccharide Synthesis The requirement f o r i s o p r e n o i d l i p i d s f o r exopolysaccharide synthesis i s a l s o common to other repeating u n i t - c o n t a i n i n g glycan polymers l o c a t e d e x t e r n a l to the c e l l membrane i . e . the same c a r r i e r l i p i d s are used f o r synthesis of peptidoglycan, t e i c h o i c a c i d s , l i p o p o l y saccharide and exopolysaccharides. Considerable i n d i r e c t evidence suggests that the a v a i l a b i l i t y of i s o p r e n o i d l i p i d phosphate i s one of the most c r i t i c a l f a c t o r s a f f e c t i n g exopoly saccharide synthesis (24). Any mutation a f f e c t i n g i s o p r e n o i d l i p i d synthesis w i l l thus a f f e c t exopolysaccharide production. Various authors have i n d i c a t e d that b a c t e r i a c o n t a i n 6.5-20 mg i s o p r e n o i d l i p i d % dry weight ( c a l c u l a t e d from r e s u l t s i n 25,26). I t has a l s o been suggested that i t s a v a i l a b i l i t y could be c o n t r o l led through phosphorylation of the f r e e a l c o h o l and dephosphory-


77, G O

Ο*

§

^

oo r~~

£i

c? &

=i.


U

Table 1.

Location of Sugar Transferase A c t i v i t i e s

72

Ο

68

3

Cytoplasmic

Outer membrane

membrane

81

lOO*

75

100*

Gal Transfer (%)

Spheroplast membrane

Crude membrane

G l c - l - P Transfer (%)

techniques.

* A c t i v i t i e s were of the order of 0.154 nmol/mg p r o t e i n / h and 0.282 nmol r e s p e c t i v e l y .

ci

η

£J£. aeroqenes type 8, G l c - l - P and Gal I + I I t r a n s f e r a s e s assayed by standard

^


is

CO

I

2. 8CO

Ci

ο "Ρ

Ci 3 ft


48

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

l a t i o n of the a l c o h o l phosphate and pyrophosphate (27). Unfortu n a t e l y , Gram negative b a c t e r i a do not take up mevalonic a c i d and i t i s not p o s s i b l e to l a b e l the l i p i d precursors and thus o b t a i n more accurate e s t i m a t i o n of the amount present i n c e l l s than can be found from d i r e c t e x t r a c t i o n . However, one p o s s i b l e way of i n c r e a s i n g the i s o p r e n o i d l i p i d content appeared to be through s e l e c t i o n f o r b a c i t r a c i n r e s i s t a n c e , s i n c e t h i s a n t i b i o t i c binds very s t r o n g l y to i s o p r e n o i d l i p i d s and e f f e c t i v e l y removes them from b i o s y n t h e t i c processess. Mutants with c o n s i d e r a b l y elevated b a c i t r a c i n r e s i s t a n c e have been i s o l a t e d i n our l a b o r a t o r y and some undoubtedly y i e l d more exopolysaccharide and show increased t r a n s f e r of monsaccharides to l i p i d . (Other mutants were l i t t l e d i f f e r e n t from w i l d type i n a l l respects tested or had l o s t the a b i l i t y to synthesize exopolysaccharide.) I t i s a l s o p o s s i b l e that some mutants d e f e c t i v e i n p e p t i d o glycan synthesis might r e q u i r e l e s s i s o p r e n o i d l i p i d than w i l d type c e l l s , thus r e l e a s i n g more f o r exopolysaccharide s y n t h e s i s . A mutant of t h i s type has r e c e n t l y been i s o l a t e d from E. c o l i Β and, u n l i k e the parent b a c t e r i a , produces exopolysaccharide (R.W. North, unpublished r e s u l t s ) . S i m i l a r observations have a l s o been reported during attempts to prepare mutants f o r genetic engineering. The reverse s i t u a t i o n , reduced i s o p r e n o i d l i p i d content, i s a l s o d i f f i c u l t to study and can only be checked i n d i r e c t l y . Mutants with l e s s l i p i d than w i l d type b a c t e r i a have not been c h a r a c t e r i z e d , but a group of CR (crenated) mutants i s o l a t e d from E. aerogenes have c h a r a c t e r i s t i c s which i n d i c a t e that they may be c o n d i t i o n a l mutants of t h i s type (28). These b a c t e r i a have rough c o l o n i a l appearance at lowered i n c u b a t i o n temperature and t h i s has been a s c r i b e d to a reduced content of l i p o p o l y s a c c h a r i d e . Exopolysaccharide i s not synthesized u n t i l growth has ceased. The enzymes f o r p o l y s a c c h a r i d e synthesis are present i n the b a c t e r i a grown a t low temperature and on t r a n s f e r to washed c e l l suspensions ( n o n - p r o l i f e r a t i n g c o n d i t i o n s ) exopolysaccharide i s immediately formed i n the presence or absence of chloramphenical. Thus no new enzymes have to be formed but at low temperature the synthesis of peptidoglycan - e s s e n t i a l f o r c e l l v i a b i l i t y appears to take precedence over exopolysaccharide production and, to a l e s s e r extent l i p o p o l y s a c c h a r i d e s y n t h e s i s . At 37°C, the mutants are i d e n t i c a l i n a l l respects t e s t e d to w i l d type b a c t e r i a . The mutants are not l i k e c l a s s i c a l membrane mutants, d e f i c i e n t i n membrane p h o s p h o l i p i d and s u s c e p t i b l e to various detergents. S i m i l a r c h a r a c t e r i s t i c s were observed i n a polysaccharide-forming pseudomonad (29). The e x t r a c e l l u l a r polymer was only produced l a t e i n the l o g phase of growth and i n the s t a t i o n a r y phase, having s e v e r a l of the a t t r i b u t e s of a secondary m e t a b o l i t e . Could t h i s too be due to i n s u f f i c i e n t i s o p r e n o i d l i p i d i n the growing and peptidoglycan-forming b a c t e r i a ?



4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

49

In the l i t e r a t u r e , frequent r e p o r t s of exopolysaccharide production being favoured by growth a t low temperature a r e to be found; a l t e r n a t i v e l y the polymer i s s a i d to be a product of the c e l l s a f t e r growth has ceased. No s a t i s f a c t o r y explanation f o r these observations has been provided, y e t b a c t e r i a from the l o g or e a r l y s t a t i o n a r y phases of growth appear to produce exopolysaccharide i n washed suspension a t s i m i l a r r a t e s . T h i s could be due to l i m i t a t i o n of exopolysaccharide synthesis during a c t i v e growth through the a v a i l a b i l i t y of i s o p r e n o i d l i p i d ; i t would be needed f o r the formation of w a l l polymers u n t i l l a t e i n the l o g phase of growth. L i m i t a t i o n of c a r r i e r l i p i d a l s o occurs i n c e r t a i n Salmonella mutants d e f e c t i v e i n l i p o p o l y s a c c h a r i d e formation. Mutants forming the l i p i d - l i n k e d O-antigen but unable to t r a n s f e r i t to the appropriate acceptor have been c h a r a c t e r i z e d (30). Thus p a r t of the normal i s o p r e n o i d l i p i d i s no longer a v a i l a b l e f o r other processess, e f f e c t i v e l y reducing the t o t a l present i n the bacteria. Mutants of t h i s type could not produce exopolysaccharide although others d e f e c t i v e i n l i p o p o l y s a c c h a r i d e synthesis but not accumulating l i p i d - l i n k e d glycans, had t h i s c a p a c i t y (31). Several d i f f e r e n t types of mutations can thus a f f e c t i s o p r e noid l i p i d a v a i l a b i l i t y and consequently exopolysaccharide production. These are summarized i n F i g . 6. The i n d i r e c t evidence suggests a d i s t i n c t s e r i e s of p r i o r i t i e s f o r i s o p r e n o i d l i p i d utilization. The e s s e n t i a l w a l l polymer peptidoglycan has p r i o r i t y over l i p o p o l y s a c c h a r i d e which i n turn has p r i o r i t y over exopolysaccharide synthesis ( F i g s . 7 and 8 ) . T h i s could to some extent be achieved through s p a t i a l s e p a r a t i o n of the polysaccharide s y n t h e s i z i n g systems w i t h i n the m i c r o b i a l membrane but obviously requires further elucidation. The f i n a l stages - m o d i f i c a t i o n and e x t r u s i o n As already discussed, the o l i g o s a c c h a r i d e r e p e a t i n g u n i t s accumulate on the c a r r i e r l i p i d and t h i s type of mechanism probably a p p l i e s to a l l exopolysaccharides other than dextrans, levans and r e l a t e d polymers (24). The mechanism could accommodate b a c t e r i a l a l g i n a t e synthesis i f i t i s regarded i n i t i a l l y as a homopolymer of Dmannuronic a c i d and i s probably a l s o v a l i d f o r the glucans secreted by Agrobacterium species. However, many exopolysaccharides c o n t a i n a c y l and k e t a l s u b s t i t u e n t s . Are these added while the repeating u n i t s are attached to l i p i d or at some l a t e r stage? (Fig.9). P r e l i m i n a r y evidence suggests that a c y l a t i o n occurs while the o l i g o s a c c h a r i d e i s s t i l l attached to the l i p i d , but f u r t h e r s t u d i e s are needed. This might i n d i c a t e the lower degree of p y r u v y l a t i o n o c c u r r i n g i n polysaccharide produced a t higher growth r a t e s (and higher r e s u l t a n t l i p i d turnover rates) reported i n some s p e c i e s . The carbon source probably has no d i r e c t e f f e c t (Table 2). Considerable v a r i a t i o n s i n a c y l a t i o n are found w i t h i n a s i n g l e polysaccharide. Thus, a c e t y l groups may occur on each r e p e a t i n g u n i t or on every second repeating u n i t i n one E. aero gene s


50

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Wild-type bacteria (lipopolysaccharides, capsules, slime)

/ i

CR mutants* (decreased lipopolysaccharides, no slime or capsule until growth ceases)

SL mutants (lipopolysaccharides, slime)

\ Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch004

Ο mutants (lipopolysaccharides)

« • Bacitracin-resistance

CRO mutants (decreased lipopolysaccharides)

SR mutants (1 repeat unit of lipopolysaccharide side chain)

R mutants* (core lipopolysaccharides, side chains unattached)

R mutants (inner core only)

* Mutations affecting isoprenoid lipidsfdirectly or indirectly) Biochemical Society Transactions

Figure 6.

How mutations affect the production of exopolysaccharides (31)

\

GROWING

CELLS

E N D OF L O G P H A S E -

Figure 7.

EXOPOLYSACCHARIDE

OR L O W I N C U B A T I O N

TEMPERATURE

Carrier lipid utilization


Microbial

SUTHERLAND

IPP-

C 5 5 - ISOPRENYL

IPA -

C 5 5 - ISOPRENOID ALCOHOL

PYROPHOSPHATE

ISOPENTENYL

1 1


Exopolysaccharide

IP-

PYROPHOSPHATE +

51

Synthesis

C - I S O P R E N Y L PHOSPHATE 5 5

FARNESYL

PYROPHOSPHATE

POLYMER -

MUCOPEPTIDE LPS orTEICHOIC ACID EXOPOLYSACCHARIDE

INTRACELLULAR and MEMBRANE - BOUND PRECURSORS

IPA

Figure 8.

Regulation of carrier lipids

L I P I D - P - P - GIc-GIc I Man I GIcA I Man • A c e t y l CoA

[GIC- Glc] I Man I GIcA I Man

n

OR

• A c e t y l CoA

+ PEP

I

• PEP

r

1

L I P I D - P - P - G I c - GIc [Glc-GlcJ I I Man-O-Ac Man-O-Ac I I GIcA GIcA I I Man = Pyr Man = Pyr Figure 9. Possible exopolysaccharide acylation mechanisms n


In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977. 1.92 1.74

5.48

3.61 3.67

14.5

72.8

Galactose

17.1

79.4

78.8

Sodium pyruvate

Sodium succinate

18.5

18.0

74.0

Raffinose

3.40

6.10 6.22

3.46 3.52

1.30

1.55

0.60

6.96

3.90

2.65 2.87

6.28 5. 81

3.75

16.9

73.6

Sucrose

Maltose

4.03

17.4

79.7

Lactose

1.89

3.78

5.20

5.36

3.16

16.7

75.7

17.9

72.1

Ribose

1.14 5. 70

3.46

16.5

70. Ο

Arabinose

8.75

3.36

0.43

2.20

6.46

3.84

18.2

74.4

Rhamnose

15.8

67.3

Xylose

3.39

6.64

Fructose

3.37

18.5 15.8

74.0

69.4

Mannose

3.23

5. 55 6.32

3.52

15.2

75. Ο

Exopolys acc har ide mg/ml

Glucose

Carbon Source

Pyruvic A c i d %

of

r

Acetate %

R e s u l t s

of a Pseudomonas Exopolysaccharide Derived from Growth on Various Substrates ^ Williams, 1974)

Deoxyhexose %

The Composition

Hexose %

Table 2.


Ω Ω

>

F *d Ο F *

>

W

ο

§

M F F

> Ω

αϊ to

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

53


s t r a i n (32). I t has a l s o been demonstrated that a c e t y l a t i o n can be l o s t from a s t r a i n without l o s s of exopolysaccharides y n t h e s i z i n g capacity (33) . In c o n t r a s t , loss of any enzyme c o n t r i b u t i n g to the polysaccharide s t r u c t u r e would lead to a non-mucoid v a r i a n t . S i m i l a r l y , p y r u v y l a t i o n a l s o appears i n e s s e n t i a l f o r polysaccharide synthesis as, under c e r t a i n growth c o n d i t i o n s pyruvate groups can be l o s t but polysaccharide of apparently normal carbohydrate composition produced. The exopolysaccharides studied so f a r , have mainly comprised repeating u n i t s with a s i n g l e attached monosaccharide s i d e - c h a i n . I t i s p o s s i b l e that c o n s t r u c t i o n of the longer s i d e chains- found i n xanthan gum or c o l a n i c a c i d might r e q u i r e some other mechanism such as c o n s t r u c t i o n of main-chain and s i d e - c h a i n u n i t s on separate c a r r i e r l i p i d s p r i o r to assembly. (An analogy can be found i n l y s o g e n i c conversion, 34.) The mode of f i n a l r e l e a s e from the i s o p r e n o i d l i p i d has not yet been demonstrated. I t i s u n l i k e l y that the process occurs through non-enzymic r e l e a s e of the i n c r e a s i n g l y h y d r o p h i l i c elongating polysaccharide chain. T h i s would probably leave the c a r r i e r l i p i d u n a v a i l a b l e f o r f u r t h e r polysaccharide s y n t h e s i s . In capsuleproducing s t r a i n s , a l i g a s e r e a c t i o n may remove the polymer chain and attach i t to the c e l l s u r f a c e . It is unlikely that h y d r o l y s i s of the polysaccharide chain occurs at t h i s stage unless a h i g h l y s p e c i f i c enzyme cleaves the t e r m i n a l , phosphate l i n k e d monosaccharide: L i p i d - Ρ - Ρ - Glucose - Galactose

etc.

Enzymes reducing the degree of polymerization have been i d e n t i f i e d i n alginate-producing b a c t e r i a (35) but the f u n c t i o n of the enzyme i s probably unconnected with polymer r e l e a s e of t h i s type. Mutants unable to attach to the c e l l surface (SI mutants) have been widely found, presumably through l o s s of the capsule a t t a c h ment s i t e s on the c e l l s u r f a c e ; other micro-organisms always produce exopolysaccharide as e x t r a c e l l u l a r s l i m e . The chain length of the polymer may a l s o depend on the growth rate i n a manner analogous to l i p o p o l y s a c c h a r i d e side-chains (36), but t h i s needs f u r t h e r study. Higher growth r a t e might lead to more r a p i d turnover of the c a r r i e r l i p i d and r e l e a s e of polymer of lower molecular weight. This i s obviously important to the commercial producer. I t may a l s o be advantageous to use rough mutants ( i . e . s t r a i n s with surface defects) which autoagglutinate of f l o c c u l a t e and lead to e a s i e r polymer recovery. Thus exopoly saccharide production should be examined along with the synthesis of other polysaccharides and not i n i s o l a t i o n .


EXTRACELLULAR


54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Hexokinase Phosphoglucomutase Phosphoglucose Isomerase Phosphomannose Isomerase Phosphomannomutase UDP-Glc Pyrophosphorylase GDP-Man Pyrophosphorylase UDP - Gal Epimerase UDP-Glc Dehydrogenase Glc Transferase I Glc Transferase 1 Man Transferase I Man Transferase Π GIcA Transferase Polymerase (s) Ketalase Acetylase Figure 10.

MICROBIAL

POLYSACCHARIDES

- [Glc - Glc] | Man - Ο - Ac QJ ^ J_ _ ipc Man « ryr ^ ^ ^* / \ \ • ' / _ \ \ " GDP-Man UDP - Glc A « j UDP- Glc ^ UDP-Gal F7 T* Man-I-P Glc-l-P N

C

%

Î5

Man-6-P T4

T*

Glc-6-P TL

Fruct-6-P J3

Biosynthesis of Xanthomonas polysaccharides

REACTION

CONTROL

Substrate

Substrate entry

Membrane CYTOPLASM

Hexose-phosphate

Hexose-phosphate level

X D P - hexose

XDP-hexose pyrophosphorylase X D P - hexose hydrolase

i

Lipid - Ρ - Ρ - hexose Isoprenoid lipid availability Lipid-P - Ρ - oligosaccharide

Ψ Polysaccharide Figure 11.

?

The control of ρ dysaccharide synthesis


4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

55


Summary The production of m i c r o b i a l exopolysaccharides i n v o l v e s a r e l a t i v e l y l a r g e number of enzymes, some of which are i n v o l v e d i n the formation of other polysaccharides while others are unique to exopolysaccharide s y n t h e s i s . By e x t r a p o l a t i o n from r e s u l t s obtained with other s p e c i e s , a b i o s y n t h e t i c pathway f o r X. campestris polysaccharide can be constructed ( F i g . 10). Loss of most of these enzymes leads to l o s s o f polysaccharide product i o n , but v a r i a t i o n s i n a c y l a t i o n or k e t a l a t i o n occur and may be of importance to the i n d u s t r i a l m i c r o b i o l o g i s t . C o n t r o l of p o l y s a c c h a r i d e synthesis probably occurs at a number of l e v e l s ( F i g . 11), and some mutations with a l t e r e d p o l y s a c c h a r i d e r e g u l a t i o n may have advantageous p r o p e r t i e s .

Literature Cited 1. Gibbons, R.J. and Nygaard, M. Arch. oral Biol., 13, 12491249 (1968). 2. Smith, E.E.

FEBS Letters, 12, 33-37 (1970).

3. Herbert, D. andKornberg,H.L. Biochem. J., 156, 477-480 (1976). 4. Roseman, S. In'MetabolicPathways'Ed. Hokin, L.E., 6, 41-89 (1972). Academic Press, London and New York. 5. Preiss, J . In'CurrentTopics in Cellular Regulation' 1, pp. 125-160 (1969). 6. Grant, W.D., Sutherland, I.W. and Wilkinson, J.F. J . Bact., 103, 89-96 (1970). 7. Bernstein, R.L. and Robbins, P.W. J . Biol. Chem., 240, 391-397 (1965). 8. Kornfeld, R.H. and Ginsburg, V. Biochim. Biophys. Acta, 117, 79-87 (1966). 9.

Ward, J.B. and Glaser, L. Biochem. Biophys. Res. Commun., 31, 671-6 (1968).

10. Ward, J.B. and Glaser, L. Arch. Biochem., 134, 612-622 (1969). 11.

Lieberman, M.M. and Markovitz, A. J . Bact., 101, 965-972 (1970).


56

12.

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Lieberman, M.M., Shaparis, A. and Markovitz, A. J. Bact., 101, 959-964 (1970).


13. Markovitz, A. In 'Surface Carbohydrates of Prokaryotes', Ed. Sutherland, I.W., Academic Press, London and New York (In press). 14.

Lawson, C.J., McCleary, C.W., Nakada, H.I., Rees, D.A., Sutherland, I.W. and Wilkinson, J.F. Biochem. J., 115, 947-958 (1969).

15.

Sutherland, I.W. Biochem. J., 115, 935-945 (1969).

16.

Troy, F.A., Frerman, F.A. and Heath, E.C. 246, 118-133 (1971).

17.

Sutherland, I.W. and Norval, M. Biochem. J., 120, 567-576 (1970).

18.

Sandermann, H. and Strominger, J.L. J . Biol. Chem., 247, 5123-5131 (1972).

19.

Poxton, I.R., Lomax, J.A. and Sutherland, I.W. J . Gen. Microbiol., 84, 231-233 (1974).

20.

Lomax, J.Α., Poxton, I.R. and Sutherland, I.W. FEBS Letters 34, 232-234 (1973).

21.

Osborn, M.J., Gander, J.E. and Parisi, E. J . Biol. Chem.,

J . Biol. Chem.,

247, 3973-3986 (1972). 22.

Mayer, R.M.

23.

Lennarz, W.J. and Scher, M.G. Biochim. Biophys Acta, 265, 417-441 (1972). Sutherland, I.W. In"SurfaceCarbohydrates of Prokaryotes", pp. - , Academic Press, London and New York (In press).

24.

Bio chim. Biophys. Acta, 252, 39-47 (1971).

25.

Umbreit, J.N., Stone, K.J. and Strominger, J.L. J . Bacteriol. 112, 1302-1305 (1972).

26.

Dankert, M., Wright, Α., Kelley, W.S. and Robbins, P.W. Arch. Biochem., 116, 425-435 (1966).

27.

Willoughby, E . , Higashi, Y. and Strominger, J.L. J . Biol. Chem.,247, 5113-5115 (1972).


4.

28.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

57

Norval, M. and Sutherland, I.W. J . Gen. Microbiol., 57, 369-377 (1969)

29.

Williams, A. Ph.D. Thesis, University College, Cardiff. (1974)


30.

Kent, J.L. and Osborn, M.J. Biochemistry, 7, 4396-4408 (1968). 31. Sutherland, I.W. Biochem. Soc. Trans., 3, 840-843 (1975). 32. Sutherland, I.W. In "Surface Carbohydrates of Prokaryotes", pp. - , Academic Press, London and New York, (In press). 33.

Garegg, P.J., Lindberg, Β., Onn, T. and Holme, T. Acta Chem. Scand., 25, 1185-1194 (1971).

34.

Wright, A. J . Bacteriol., 105, 927-936 (1971).

35.

Madgwick, J., Haug, A. and Larsen, B. Acta Chem. Scand., 27, 711-712 (1973). Collins, F.M. Aust. J . Exp. Biol. Med., 42, 255- 2 (1964).

36.


Extracellular Microbial Polysaccharides

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