Extracellular Microbial Polysaccharides

1 Culture Maintenance and

Productivity

DENIS K. KIDBY

Downloaded by 80.82.77.83 on June 4, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch001

Department of Soil Science and Plant Nutrition, The University of Western Australia, Nedlands, Western Australia, 6009

Microbial productivity is based upon a very large store of genetic information. In a typical bacterial cell, there are more than one million items encoded. At the i n i t i a t i o n of inoculum build-up, i t is a common practice to transfer approximately 10 cells to a fresh medium. To retain the complete genetic identity of such an inoculum, for even a single generation, 10 base pairings must occur with complete f i d e l i t y . However, examination of such a c e l l population would reveal that thousands of errors had occurred. The f i d e l i t y of DNA replication is nevertheless impressive, and given s k i l f u l management, microbes can approach the r e l i a b i l i t y of solution chemistry in terms of product reproducibility. While genetic change may be a disaster when uncontrolled, i t is also the means of improving productivity. Genetic alterations were once achieved more or less by chance. However, the possibility now exists for the deliberate, and specific, alteration of genotype to yield productive chimeras limited only by the imagination. One can envisage the real possibility of producing a bacterial c e l l which could extract i t s energy and growth requirements from a few simple salts, the a i r and sunlight, producing a bacterial product such as Xanthan Gum or, an algal product such as agarose. However, despite such advances in the manipulation of genes, i t seems certain that the inherent genetic i n s t a b i l i t y of microbes w i l l remain an important problem for many years; and it is largely to this type of d i f f i c u l t y that the present paper is addressed. Before discussing i n s t a b i l i t y , the origins of industrial cultures w i l l be briefly considered. 9

16

Sources of Microbes Natural Sources. Many useful microbes are directly obtainable from the s o i l or other natural sources. It is often possible to employ unusual or extreme conditions as selective agents in the search for microbes with special a b i l i t i e s . Bacteria isolated from hot springs, can be grown near the temperature of boiling water (1). Acid mine leachings harbour 1

Sandford and Laskin; Extracellular Microbial Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


2

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

b a c t e r i a able to grow at high concentrations of s u l f u r i c a c i d (2). Microbes f r e e of toxins or e s p e c i a l l y a l l e r g e n i c substances may be sought i n f o o d s t u f f s i n which they are known to r e g u l a r l y occur i n h i g h c o n c e n t r a t i o n s . I s o l a t i o n Procedures. The p r i n c i p l e s employed are those of s e l e c t i v e enrichment or i n h i b i t i o n . The r e q u i r e d , or suspected, n u t r i t i o n a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the organism sought w i l l d i c t a t e and a c t u a l procedure. The o x i d a t i o n , r e d u c t i o n , b i n d i n g , or r e l e a s e , of dyes are p a r t i c u l a r l y adapt a b l e f o r s e r v i c e as i n d i c a t o r s of s p e c i f i c biochemical events. The possession of a p a r t i c u l a r enzyme, or s e r i e s of enzymes, may be l i n k e d to e i t h e r the a b i l i t y , or i n a b i l i t y , to grow on a p a r t i c u l a r medium. B i o l o g i c a l i n d i c a t o r s such as the growth of an i n d i c a t o r organism are p a r t i c u l a r l y s e n s i t i v e to such funct i o n s as the e x c r e t i o n of vitamins or amino a c i d s . Ingenious methods have been devised f o r the s e l e c t i o n of c h a r a c t e r i s t i c s which are by t h e i r nature c r y p t i c and seemingly i n a c c e s s i b l e f o r s e l e c t i o n . For example Okanishi and Gregory Ô ) were able to devise a simple method to r e v e a l yeast c o l o n i e s possessing higher than normal methionine l e v e l s . Protocols f o r the i s o l a t i o n of s p e c i f i c n u t r i t i o n a l types may be sought i n the taxonomic l i t e r a t u r e (4, 5). Specific procedures f o r various groups of organisms are a v a i l a b l e i n the recent l i t e r a t u r e (6>, J7> 8). However, the seeker of d e s i r a b l e microbes must o f t e n r e l y upon h i s own r e s o u r c e f u l n e s s . A fairly thorough biochemical understanding of the event of i n t e r e s t can b e a most u s e f u l guide to i s o l a t i o n procedures. In the case of e x t r a c e l l u l a r products, such as polysaccha r i d e s , there may or may not be c h a r a c t e r i s t i c a l l y mucoid colonies. S e l e c t i v e procedures should, i f p o s s i b l e , e x p l o i t some s p e c i f i c property of the d e s i r e d p o l y s a c c h a r i d e . However, there are p o s s i b i l i t i e s f o r i n d i r e c t s e l e c t i o n using a s s o c i a t e d c h a r a c t e r i s t i c s . For example, many c h a r a c t e r i s t i c s , s u i t e d to r e p l i c a - p l a t i n g methods, are a s s o c i a t e d with polysaccharide producing Xanthomonas campestris ( 9 ) . In the case of mucoid E s c h e r i c h i a c o l i , there appears to be a s s o c i a t e d UV s e n s i t i v i t y (10) . R e p l i c a - p l a t i n g procedures are f r e q u e n t l y the most u s e f u l technique s i n c e one can s e l e c t f o r c e l l s which e i t h e r grow or do not grow. D i a g n o s t i c procedures which are d e s t r u c t i v e may a l s o be used since a l l m a t e r i a l under i n v e s t i g a t i o n i s r e t a i n e d on the r e p l i c a s . The employment of s p e c i f i c enzymes f o r the recogn i t i o n of c e r t a i n types of polysaccharides i s an i n t e r e s t i n g p o s s i b i l i t y f o r the development of screening programmes. In t h i s connection i t i s i n t e r e s t i n g to note that r e c o g n i t i o n systems based upon enzyme s p e c i f i c i t y may already occur i n bacteriophage (11) . C u l t u r e C o l l e c t i o n s . Searching f o r microbes i n e x i s t i n g c u l t u r e s w i l l f r e q u e n t l y be quicker, cheaper and e a s i e r than i s o l a t i o n from nature. As an a i d to such a search, H e s s e l t i n e and Haynes (12) have w r i t t e n a guide to c o l l e c t i o n s containing


1.

KiDBY

Culture

Maintenance

and

i n d u s t r i a l l y u s e f u l microbes. However, there can be no f o r thorough searching of the current l i t e r a t u r e .


3

Productivity

substitute

Maintenance of Genotype Nature of the Problem. An i n d u s t r i a l l y u s e f u l microbe i s an asset which may range from being moderately valuable to almost p r i c e l e s s . The p r e s e r v a t i o n of such an asset deserves a p r i o r i t y which i t seldom r e c e i v e s . The greatest b a r r i e r to s u c c e s s f u l p r e s e r v a t i o n of genotype may be a f a i l u r e to appreciate that: ( i ) microbes are i n h e r e n t l y unstable, ( i i ) there i s no method yet devised f o r the complete p r e s e r v a t i o n of genotype. Inherent I n s t a b i l i t y of Microbes. The p o t e n t i a l f o r genotype v a r i a b i l i t y has been i n d i c a t e d i n the i n t r o d u c t o r y remarks. I t i s now necessary to discuss the a c t u a l mechanism of change and how these r e l a t e to phenotype. A l l regions of a gene are mutable. Some genes are more mutable than others because they have i n t r a g e n i c regions of high m u t a b i l i t y , are i n f l u e n c e d by some other gene which i s i t s e l f mutable or, are under the c o n t r o l of genes which promote mutation. A l l of these mechanisms are known to occur, i n c l u d i n g some i n which the m u t a b i l i t y i s e f f e c t e d by an extrachromosoma1 element o r , an i n f e c t i o u s agent (13). I t i s these more h i g h l y mutable genes, and e s p e c i a l l y those cases i n v o l v i n g i n f e c t i o u s agents, that are most troublesome. Mutations may be e i t h e r r e p l i c a t i o n dependent or r e p l i c a t i o n - i n d e p e n d e n t . I t i s speculated (14) that replication-dependent mutations r e f l e c t e r r o r s i n DNA r e p l i c a t i o n , and replication-independent mutations r e f l e c t error-prone r e p a i r systems, Mutations may i n v o l v e : ( i ) frame-shift; ( i i ) deletion; ( i i i ) i n s e r t i o n ; ( i v ) base p a i r s u b s t i t u t i o n . The e f f e c t on the code may be e i t h e r the production of missense, nonsense, or a non-code f u n c t i o n may be l o s t . The r e s u l t i n g phenotypes may i n c l u d e : ( i ) a l t e r e d RNA base sequence; ( i i ) a l t e r e d amino a c i d sequence; ( i i i ) premature termination; ( i v ) degenerate s i l e n c e . A c e r t a i n p r o p o r t i o n of these mutants w i l l be c r y p t i c , p a r t i c u l a r l y those i n v o l v i n g missense. Mutations which lead to the i n s e r t i o n of a s i m i l a r amino a c i d o r , because of code degeneracy, the w i l d type amino a c i d , w i l l u s u a l l y not be revealed. I t has been c a l c u l a t e d that 25% of 549 base p a i r s u b s t i t u t i o n s i n v o l v e degeneracy (15) . I t i s a l s o i n t e r e s t i n g to note that there i s a greater than random p r o b a b i l i t y that base p a i r s u b s t i t u t i o n s w i l l lead to s u b s t i t u t i o n of a s i m i l a r r a t h e r than a d i s s i m i l a r amino a c i d (16). L e t h a l mutations w i l l a l s o be c r y p t i c since these w i l l not p e r s i s t , unless they are c o n d i t i o n a l . Intragenic mutations are non-random. S i t e s which are h i g h l y mutable are hot spots (17) . Evidence on the nature of hot spots has been reviewed by Clarke and Johnston (1976) and w i l l be merely summarized here. High M u t a b i l i t y Regions, ( i ) Frameshift mutations tend to occur i n regions of repeated base p a i r s . Runs of e i t h e r AT or GC base p a i r s have been a s s o c i a t e d with f r a m e s h i f t s . ( i i ) Base



4

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MICROBIAL

POLYSACCHARIDES

p a i r s u b s t i t u t i o n s are i n f l u e n c e d by neighbouring bases. The AT-GC s u b s t i t u t i o n induced by 2-aminopurine at the second p o s i t i o n of a t r i p l e t has been demonstrated to occur 23 times more f r e quently when an AT base p a i r was present i n the t h i r d p o s i t i o n (18). ( i i i ) Mutator polymerase acts p r e f e r e n t i a l l y on s p e c i f i c regions of the gene, ( i v ) The frequency and l o c a t i o n of d e l e t i o n s i s non-random and such s i t e s are considered d e l e t i o n hot spots, (v) U l t r a - v i o l e t induced mutations are most frequent i n t r a c t s of p y r i m i d i n e s . Development of a S t a b l e Mutation. Most mutations are formed from pre-mutational l e s i o n s . The l e s i o n may or may not be r e p a i r e d o r , the r e p a i r process i t s e l f may lead d i r e c t l y to mutat i o n . F a i l i n g r e p a i r , the pre-mutational l e s i o n may be e s t a b l i s h ed as a mutation by DNA r e p l i c a t i o n . The events i n v o l v e d i n development of a mutation are summarized i n Figure 1. Any one of these steps may be subject to the i n f l u e n c e of adjacent base pairs. In the l i g h t of these o b s e r v a t i o n s , one might ask what avenues e x i s t f o r the a m e l i o r a t i o n or removal of hot spots? I f the mutation i s e f f e c t e d by a mutagen, i t may be p o s s i b l e to e i t h e r remove or suppress the c o n d i t i o n l e a d i n g to the presence of the mutagen or n e u t r a l i z e i t s a c t i v i t y with an antimutator. Precedents f o r t h i s l a t t e r approach are now w e l l documented (14). Antimutagenesis. I t has been q u i t e p r o p e r l y s t a t e d (14) that one cannot understand mutagenesis or the r e g u l a t i o n of mutation frequency without c o n s i d e r i n g antimutagenic e f f e c t s . Antimutagenesis may be d e f i n e d as a decrease i n the a c t u a l r a t e of mutation. Decreased apparent rates may be caused by e i t h e r a l t e r e d s u r v i v a l or dose r e d u c t i o n , and these e f f e c t s are termed apparent antimutagenesis. A mutation or premutation may a r i s e by: ( i ) r e a c t i o n between a mutagen and DNA; ( i i ) i n c o r p o r a t i o n of a mutagen-altered precursor or base analogue; ( i i i ) r e p l i c a t i o n e r r o r ; ( i v ) recombination e r r o r ; (v) r e p a i r e r r o r ; ( v i ) t r a n s c r i p t i o n e r r o r ; ( v i i ) t r a n s l a t i o n e r r o r . The l a s t two mechanisms i n v o l v e the p r o d u c t i o n of error-prone RNA or p r o t e i n s which a l t e r the base sequence of DNA e i t h e r d i r e c t l y or i n d i r e c t l y (19, 20, 21). C l a r k e and Shankel (14) have d i s t i n g u i s h e d between genetic antimutagenesis, which i s the antimutagenic e f f e c t of r e p l i c a t i o n genes, r e p a i r genes, or other genetic determinants, and p h y s i o l o g i c a l antimutagenesis which i s achieved by added chemicals or a l t e r e d c e l l c o n d i t i o n s . The p h y s i o l o g i c a l mechanism would appear to o f f e r c o n s i d e r a b l e p o t e n t i a l f o r the r e d u c t i o n of mutation r a t e s f o r c e r t a i n c l a s s e s of mutation. For example, adenosine appears to be capable of v i r t u a l l y a b o l i s h i n g the mutagenicity of purine mutagens (14). Spontaneous mutation rates have a l s o been d r a m a t i c a l l y reduced by the use of a c r i d i n e s (22). An o b s e r v a t i o n of c o n s i d e r a b l e i n t e r e s t i s that genes are more l i k e l y to mutate when being t r a n s c r i b e d (14). Thus the r e p r e s s i o n of gene a c t i v i t y i s antimutagenic. I t might be expected,


1. KiDBY

Culture

Maintenance

and


WILD GENOTYPE

WILD PHENOTYPE

ι

5

Productivity

LESION REPAIR

PREMUTATION

SUPRESSION SILENT MISSENSE DEGENERACY

REPLICATION

MUTATION SELECTION

Figure 1.

Sequences of events in mutation and selec tion



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t h e r e f o r e , that i n maintenance and inoculum build-up c u l t u r e s , the r e p r e s s i o n of the productive f u n c t i o n would help to a r r e s t v a r i a b i l i t y by decreasing the r a t e of mutation. I t may a l s o be the case that r e p r e s s i o n of product formation w i l l help prevent s e l e c t i o n against producer c e l l s . There i s some evidence (23^ 24) that product r e p r e s s i o n may be of use i n reducing v a r i a b i l i t y i n Xanthomonas campestris. There seems l i t t l e reason to doubt that DNA which i s not being t r a n s c r i b e d should be r e l a t i v e l y s t a b l e . I t would be of considerable i n t e r e s t to see i f mutations i n derepressed genes are i n f a c t p r o p o r t i o n a l to t r a n s c r i p t i o n r a t e s . I t may w e l l be that c e r t a i n microbes with high product y i e l d s are i n h e r e n t l y unstable because of high t r a n s c r i p t i o n a l a c t i v i t y . L i m i t i n g the Opportunity f o r Mutation. Mutation rates may be a f u n c t i o n of r e p a i r , r e p l i c a t i o n or t r a n s l a t i o n r a t e s , of mutagen or antimutagen c o n c e n t r a t i o n s , or of p h y s i c a l c o n d i t i o n s such as r a i s e d temperature, low water a c t i v i t y , or i c e c r y s t a l s . Whatever the c o n d i t i o n l e a d i n g to mutation, the most e f f e c t i v e p r o t e c t i o n i s to minimise the exposure of the c u l t u r e to the conducive c o n d i t i o n . The growth i n mutant numbers i s a f u n c t i o n of the number of r e p l i c a t i o n s (Table I ) . I t f o l l o w s , t h e r e f o r e , that the t o t a l number of r e p l i c a t i o n s should be minimized. If r e p l i c a t i o n - i n d e p e n d e n t mutations are taken i n t o account, then i t a l s o follows that the t o t a l residence time i n c u l t u r e should be minimized. I f , as seems to be the general case, mutation i s p r o p o r t i o n a l to t r a n s l a t i o n a l a c t i v i t y , then the productive f u n c t i o n should be repressed u n t i l needed. The e x c l u s i o n or r e d u c t i o n of potent mutagens may seem too obvious to r e q u i r e f u r t h e r comment. However, many commonly o c c u r r i n g mutagens such as metal i o n s , adenine, c a f f e i n e , ozone, to name a few, seem o f t e n to escape a t t e n t i o n . The number of base analogues generated by chemical, or high temperature, treatment of concentrated sources of purine and pyrimidine bases must o f t e n be c o n s i d e r a b l e . The frequent proximity of c u l t u r e s to e l e c t r i c motors and, i n p a r t i c u l a r , atmospheres r e c e n t l y i r r a d i a t e d with u l t r a - v i o l e t l i g h t must s u r e l y produce l a r g e numbers of ozone-induced mutants. Extremely high l e v e l s of mutation have been observed i n E. c o l i exposed to as l i t t l e as 0.1 ppm ozone f o r 60«minutes (10). The question of l i m i t i n g the opportunity f o r mutation w i l l be f u r t h e r discussed i n connection w i t h p r e s e r v a t i o n techniques. L i m i t i n g the Opportunity f o r S e l e c t i o n . The s e l e c t i o n of a mutant, i n the present context, may be taken to mean the increase of any given mutant to a s i g n i f i c a n t p r o p o r t i o n of the t o t a l p o p u l a t i o n . The extent of t h i s s e l e c t i o n w i l l be a f u n c t i o n of the c u l t u r e c o n d i t i o n s and the number of generations of c u l t u r e growth permitted. S e l e c t i v e media may be employed to remove p a r t i c u l a r c l a s s e s of mutant. N u t r i t i o n a l l y r i c h media w i l l tend to preserve and o f t e n concentrate auxotrophs while a poorer medium may s e l e c t f a i r l y e f f i c i e n t l y against auxotrophs, unless



a

m = mutation r a t e

Mutant: T o t a l

Mutant C e l l s

64mN 4m

24mN 3m

8mN 2m

2mN m

0

0

16N

8N

4N

2N

Ν

Total

Cells

4

3

2

1

0

THE PROPORTION OF MUTANTS IN A GROWING CULTURE

Generations

TABLE I



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POLYSACCHARIDES

high r a t e s of c r o s s - f e e d i n g occur. Short-term P r e s e r v a t i o n . The p r e s e r v a t i o n of c e l l v i a b i l i t y f o r periods of l e s s than a few months might a r b i t r a r i l y be termed short-term p r e s e r v a t i o n . While there can be no doubt as to the d e s i r a b i l i t y of long-term p r e s e r v a t i o n , methods of a c h i e v i n g t h i s u s u a l l y provide r e l a t i v e l y i n a c c e s s i b l e i n o c u l a and, i n some cases, may be of l i m i t e d success. In order to be u s e f u l , a short -term p r e s e r v a t i o n method must provide a high recovery of v i a b l e c e l l s which grow with a minimum l a g phase. The inoculum should be e a s i l y a c c e s s i b l e and of a standard and s u i t a b l e s i z e . Subc u l t u r e to achieve v i g o r o u s l y growing and r e p r o d u c i b l e c u l t u r e s should not be necessary. I f these c r i t e r i a cannot be met, i t may be b e t t e r to consider the r o u t i n e use of i n o c u l a preserved by long-term methods. U s e f u l short-term p r e s e r v a t i o n methods are g e n e r a l l y v a r i a t i o n s of d r y i n g procedures. A p a r t i c u l a r l y s u i t a b l e method i s the d r y i n g of c u l t u r e s onto paper (2_5, 26). Paper s t r i p s have the advantage of being e a s i l y handled and are r e a d i l y adjusted i n s i z e to y i e l d an appropriate inoculum s i z e . The method has been used w i t h success f o r X. campestris NRRL B1459 ( 9 ) . Other s h o r t term p r e s e r v a t i o n methods have been reviewed elsewhere (26). The repeated t r a n s f e r of c u l t u r e s f o r r o u t i n e maintenance must be considered an unwise p r a c t i c e and i s d i f f i c u l t to j u s t i f y where a l t e r n a t i v e non-propagative methods e x i s t . Long-term P r e s e r v a t i o n . Storage of l y o p h i l i z e d , frozen, or L - d r i e d c e l l s are the p r i n c i p l e means of long-term p r e s e r v a t i o n (26). There i s an extremely widespread b e l i e f that the method of choice i s l y o p h i l i z a t i o n . This b e l i e f i s not j u s t i f i e d by e i t h e r f a c t or theory. The reasons f o r the widespread preference f o r l y o p h i l i z a t i o n are: ( i ) t h i s was the f i r s t g e n e r a l l y s u c c e s s f u l method of longterm p r e s e r v a t i o n ; ( i i ) the product has an " a t t r a c t i v e " appearance; ( i i i ) i n j u r y from concentrated solutes i n the l i q u i d s t a t e seemed a reasonable s u p p o s i t i o n ; ( i v ) p r o t e c t i o n against i n j u r y by d r y i n g at f r e e z i n g temperatures seemed an a t t r a c t i v e advantage. I t i s now c l e a r that h i g h l y concentrated solutes are not as i n j u r i o u s as has been formerly supposed and may i n f a c t exert s i g n i f i c a n t p r o t e c t i o n (27). In the l i g h t of extensive i n v e s t i g a t i o n s of the L-drying methods of Annear (28-33) by other workers (26, 34, 35), i t seems that t h i s procedure i s to be p r e f e r r e d since recovery of many d i f f i c u l t to preserve organisms i s t y p i c a l l y 10 to 100 times higher than i s achieved with l y o p h i l i z a t i o n . I t has a l s o been observed that l a r g e increases i n mutants can accompany l y o p h i l i z a t i o n (36, 3_7, 38) . While no proper comparison appears to have been made between mutant y i e l d s from l y o p h i l i z a t i o n and L - d r y i n g , i t seems reasonable to expect that the higher r e c o v e r i e s obtained by L-drying would be accompanied by l e s s damage and t h e r e f o r e fewer mutants. There are a number of steps i n p r e s e r v a t i o n and subsequent recovery procedures which may cause genetic damage ( F i g u r e 2 ) .



1.

KiDBY

Culture

Maintenance

and

Productivity

9

F r e e z i n g i s i n i t s e l f i n j u r i o u s (39) . The extent of d r y i n g a l s o appears to i n f l u e n c e the y i e l d of mutations (40, 4l)· Prophage may a l s o be induced by d e s i c c a t i o n (42, 43). The r e h y d r a t i o n procedure i s a l s o of importance and there appears to be some evidence of c e l l leakage l e a d i n g to poor recovery (27). The r e covery medium i s an important s e l e c t i v e agent and can c l e a r l y i n f l u e n c e the recovery of c e r t a i n types of mutants. For example, some medium components can i n h i b i t recovery of nonsense suppressors i n Saccharomyces c e r e v i s i a e , w h i l e other components can r e l i e v e t h i s i n h i b i t i o n (44). Storage i n the f r o z e n s t a t e has l i t t l e to recommend i t except convenience. Storage i t s e l f i s not considered to be i n j u r i o u s provided that i c e c r y s t a l damage i s precluded by h o l d i n g the temperature below -130°C (45). I t i s suggested that f o r p r e s e r v a t i o n of genotype, L - d r y i n g procedures f o r both long and short-term requirements may be found particularly successful. I t i s not c l e a r how low a temperature should be employed f o r storage of d r i e d m a t e r i a l , but i n the absence of evidence to the c o n t r a r y , as low a temperature as i s a v a i l a b l e would seem des i r a b l e . For long-term p r e s e r v a t i o n , the m a t e r i a l i s normally h e l d under vacuum while f o r short-term p r e s e r v a t i o n , l e s s s t r i n gent, and t h e r e f o r e more convenient, c o n d i t i o n s may be employed. When r e h y d r a t i n g , a low c e l l r c u l t u r e volume r a t i o should be employed. The c u l t u r e medium should be as n u t r i t i o n a l l y r i c h as i s c o n s i s t e n t w i t h good growth. This procedure w i l l to some degree s e l e c t f o r auxotrophs. However, i t i s p o s s i b l e to screen these out i n subsequent c u l t u r e i f necessary. No c e l l population i s g e n e t i c a l l y i d e n t i c a l to i t s parent c u l t u r e . The change i n i d e n t i t y can, however, be minimized by the use of methods which lead to high recovery r a t e s . The p r e s e r v a t i o n of f r e s h i s o l a t e s should not be delayed and i t i s worth adopting a standard p r o t o c o l to deal with t h i s s i t u a t i o n ( F i g u r e 3 ) . Improvement of Genotype. C o n t r o l Mutants. One of the most u s e f u l types of mutant i s the c o n t r o l mutant where feed-back i n h i b i t i o n or r e p r e s s i o n i s absent. In the case of p o l y s a c c h a r i d e production such mutants are most l i k e l y to be recognized by t h e i r production of l a r g e mucoid c o l o n i e s . C o n d i t i o n a l mutants. The c o n d i t i o n a l mutant has great potent i a l f o r c o n t r o l l i n g complex c e l l f u n c t i o n s by such simple means as r a i s i n g or lowering of temperature. Such mutants are r e l a t i v e l y easy to o b t a i n . For example, p o l y s a c c h a r i d e production which i s c o n d i t i o n a l may be switched on and o f f or, c o n d i t i o n a l growth may be switched o f f to permit polysaccharide production i n the absence of growth. C o n d i t i o n a l l y s i s i s a l s o of c o n s i d e r a b l e a p p l i c a t i o n where i t i s d e s i r a b l e , and i t u s u a l l y i s , to remove the c e l l s from the completed fermentation. L y s i s may be achieved by the i n d u c t i o n of bacteriophage. B a c t e r i o c i n s a l s o


EXTRACELLULAR

10

LYOPHILIZATION

F.EEEZJ_N6

[FREEZING!

IFREEZINGI

MICROBIAL

POLYSACCHARIDES

DRYING STORAGE

STORAGE

L-DRYING

DRYING I STORAGE

I

REHYDRATION ITHAWINGI • i GROWTH GROWTH GROWTH Figure 2. Comparisons between sequences of events involved in preservation of cells and their subsequent recovery


REHYDRATION

ENRICHMENT SELECTION

PURIFICATION

CHARACTERIZATION

REPEATED TRANSFER

VIABLE COUNT

L-DRYING

CHARACTERIZATION

Figure 3. Selection and preservation of microbes. The scheme described incorporates tests of irdried cultures to determine viability and any alteration of characteristics as a result of the preservation procedure.



1.

KiDBY

Culture

Maintenance

and

Productivity

11

o f f e r great p o t e n t i a l f o r l y s i n g of c u l t u r e s . S t a b i l i z e d Genes. The p o t e n t i a l f o r s t a b i l i z a t i o n v a r i e s according to the o r i g i n of the i n s t a b i l i t y . . In the case of hot spots, the breaking up of runs of base p a i r s might be expected to be e f f e c t i v e . An increase i n the number of genes may be e f f e c t i v e and may, i f t r a n s l a t i o n i s the r a t e - l i m i t i n g step i n production, a l s o lead to higher production l e v e l s . I t may be p o s s i b l e to t r a n s f e r genes from a r e l a t e d organism e x h i b i t i n g a more s t a b l e genotype. Stable genotypes may be f a i r l y r e a d i l y revealed by employing the s e l e c t i v e pressure of chemostat c u l t u r e (46). Methods f o r Genotype A l t e r a t i o n . Genotypes are a l t e r e d by: ( i ) induced mutation; ( i i ) spontaneous mutation; ( i i i ) t r a n s f e r of e x i s t i n g genes. The first.method i s r a p i d and some degree of s p e c i f i c i t y i s p o s s i b l e as f o r example i n the case of ozone and UV induced mutants (10). However, a l a r g e background of unwanted mutations may a l s o be present. Spontaneous mutation rates are, of course, slower, but are capable of producing the r e q u i r e d mutants i n a s u r p r i s i n g l y short time. The s e l e c t i o n pressure to o b t a i n p a r t i c u l a r types of spontaneous mutants should be a p p l i e d i n a continuous, r a t h e r than a discontinuous, manner. This permits a more complete range of p o s s i b i l i t i e s to be expressed and i s l i k e l y to lead to a more s t a b l e mutant s i n c e the d e s i r e d character can be acquired by a s e r i e s of small steps rather than one l a r g e step which could, f o r example, be due to a s i n g l e point mutation. For example, s t a b l e and high l e v e l a n t i b i o t i c r e s i s t a n c e has been achieved i n Xanthomonas by u s i n g gradient p l a t e s but was not r e a d i l y achieved when using d i s c r e t e steps (24). A p a r t i c u l a r l y h e l p f u l account of methods of mutant i s o l a t i o n i s given by Hopwood (47). Perhaps the most a t t r a c t i v e methods of genotype improvement i n v o l v e t r a n s f e r of genetic m a t e r i a l . The advantage of t h i s method i s s p e c i f i c i t y , s t a b i l i t y , and r e l a t i v e freedom from unwanted changes i n other genes. Some very e x c i t i n g a l t e r a t i o n s can be attempted by t h i s means. I t i s d e s i r a b l e f o r the organisms to be c l o s e l y r e l a t e d because the t r a n s f e r r e d gene i s more l i k e l y to behave c h a r a c t e r i s t i c a l l y i n the r e c i p i e n t . However, genes c e r t a i n l y are t r a n s f e r a b l e between d i s t a n t l y r e l a t e d species and genetic engineering may be expected to r e v o l u t i o n i z e the synthesis of n a t u r a l products. The methods of genetic t r a n s f e r among b a c t e r i a are: ( i ) conjugation; ( i i ) t r a n s d u c t i o n ; ( i i i ) t r a n s f e c t i o n ; ( i v ) t r a n s formation, and (v) i n v i t r o recombination and t r a n s f e r from divergent species or genetic engineering. The f i r s t four methods are conventional and are e x t e n s i v e l y described (48). However, genetic engineering i s a combination of methodologies and the t o t a l procedure may be v a r i e d c o n s i d e r a b l y . One r e c e n t l y described method (49) c o n s i s t s of i s o l a t i o n of the gene as i t s RNA t r a n s c r i p t i o n product, r e t r a n s c r i p t i o n back to DNA and synt h e s i s of a complementary s t r a n d . These strands are elongated w i t h homopolymer t a i l s of o l i g o - ( d G ) . This double stranded gene



12

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

i s then mixed w i t h a plasmid which has been prepared as f o l l o w s . A n i c k i s placed i n the c i r c u l a r plasmid to provide l i n e a r DNA which i s r e p a i r e d then extended w i t h a homopoIyer t a i l o f o l i g o (dC) which i s , o f course, complementary to the a r t i f i c i a l t a i l on the copied gene. The plasmid p i c k s up the gene by the complementary t a i l s e c t i o n s and, i n doing so, becomes c i r c u l a r and thus i n f e c t i v e . F o l l o w i n g i n f e c t i o n , the plasmid i s c o v a l e n t l y l i n k e d to the copied gene by host enzymes. This gene may be t r a n s f e r a b l e to a wide range o f b a c t e r i a . Furthermore, i n t h i s p a r t i c u l a r example, the gene may be removed again from the plasmid, using a s p e c i f i c r e s t r i c t i o n nuclease, and t r a n s f e r r e d to some other plasmid. Thus i t i s p o s s i b l e to conceive o f n a t u r a l products which are e i t h e r i n a c c e s s i b l e o r grown on a seasonal b a s i s , growing i n fermenters w i t h i n hours. This has considerable i m p l i c a t i o n s , not o n l y f o r production costs but f o r the r e l a t i v e ease with which production volumes can be r e g u l a t e d .

Abstract Sources of microbes and procedures for their selection, isolation and maintenance are discussed. Maintenance of genotype is considered in terms of the nature of genetic variability, antimutagenesis, inoculation schedules, growth media and preservation methods. The improvement of genotype is discussed in terms of control mutants, conditional mutants, and methods of genotype alteration. Some common practices which may be conducive to culture degeneration are discussed and suggestions are made as to alternative procedures. Literature Cited 1. Brock, T . D . , Ann. Rev. Ecology System (1970) 1, 191. 2. Lundgren, D . , et al., "Water Pollution Microbiology", John Wiley, New York (1972) 69-88. 3. Okanishi, M . , Gregory, K.F., Canad. J. Microbiol. (1970) 16, 1139. 4. "Bergey's Manual of Determinative Bacteriology" Williams and Wilkins. 5. "Abstracts of Microbiological Methods", John Wiley, New York (1969). 6. "Methods i n Microbiology" 3A, Academic Press, New York (1970) 7. "Methods in Microbiology" 3B, Academic Press, New York (1970) 8. "Methods i n Microbiology" 4, Academic Press, New York (1971) 9. Kidby, D . K . , et al., unpublished. 10. Hamelin, C., Chung, Y . S . , Mutat. Res. (1975) 28, 131. 11. Sutherland, I.W., J. gen. Microbiol. (1976) 94, 211. 12. Hesseltine, C.W., Haynes, W.C., Progress in Industrial Microbiology (1973) 12, 3. 13. Clarke, C.H., Johnston, A.W.B., Mutat. Res. (1976) 36, 147. 14. Clarke, C.H., Shankel, C.M., Bacteriol. Rev. (1975) 39, 33.


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15.


16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

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Drake,J.W.,"TheMolecular Basis ofMutation",Holden-Day, San Francisco, 1970. Vogel, F., J. Molec. Evoln. (1972) 1, 334. Benzer, S., Proc. Natl. Acad. Sci. (1961) 47, 403. Koch, R.E., Proc. Natl. Acad. Sci. (1971) 68, 773. Lewis,C.M.,Tarrant,G.M.,Mutat. Res. (1971) 12, 349. McBride, A.C., Gowans, C.S., Genet. Res. (1969) 14, 121. Talmud, P., Lewis, D., Nature (1974) 249, 563. Puglisi, P.P., Mutat. Res. (1967) 4, 289. Cadmus,M.C.,et al., Can. J. Microbiol. (1976) in press. Kidby, D.K., unpublished. Coe, A.W., Clark, S.P., Mon. Bull. Minist. Hlth. (1966) 25, 97. Lapage, S.P. et a l . , "Methods in Microbiology" 3A, Academic Press, New York, (1970) 167. Leach, R.H., Scott, W.J., J . gen. Microbiol. (1959) 21, 295. Annear, D.I., Nature (1954) 174, 359. Annear, D.I., J. Hyg. Camb. (1956) 54, 487. Annear, D.I., J. Path. Bact. (1956) 72, 322. Annear, D.I., J. Appl. Bact. (1957) 20, 17. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1958) 36, 1. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1962) 40, 1. Hopwood, D.A., Ferguson, H.M., J . appl. Bact. (1969) 32, 434. Muggleton, P.W., Progr. Ind. Microbiol. (1962) 4, 191. Hieda, Κ., Ito, T., "Freeze-drying of biological Materials" International Institute of Refrigeration, Paris (1973) 71. Webb, S.J., Tai,C.C.,Canad. J . Microbiol. (1968) 14, 727. "Cryobiology", Academic Press, N.Y. (1966) 213. Mazur, P., Science (1970) 168, 939. Webb, S.J., Nature (1967) 213, 1137. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1968) 14, 841. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 33. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 303. Queiroz, C., Biochem. Genet. (1973) 8, 85. Martin, S.M., Ann. Rev. Microbiol. (1964) 18, 1. Veldkamp, H., "Methods in Microbiology" 3A Academic Press, New York (1970) 305. Hopwood, D.A., "Methods in Microbiology" 3A Academic Press, New York (1970) 363. Hayes, W., "The Genetics of Bacteria and their Viruses" Blackwell, Oxford (1968). Rougeon, F . , Kourilsky, P., Mach, B., Nucleic Acids Res. (1975) 2, 2365.


Extracellular Microbial Polysaccharides

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