The adherence of Acidiphilium cryptum to chalcopyrite By B l a i r H e f f e l f i n g e r B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1 9 8 5 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January 1 9 9 0 © B l a i r H e f f e l f i n g e r In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree th a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date DE-6 (2/88) \ ABSTRACT A c i d i p h i l i u m cryptum i s a h e t e r o t r o p h i c a c i d o p h i l e commonly found i n a c i d i c environments and i n d u s t r i a l b i o l e a c h i n g operations. Attachment t o mineral surfaces may serve to maintain t h i s organism i n aqueous environments where i t i s subject t o removal by hydrodynamic f o r c e s . Using i n d i r e c t and d i r e c t methods we have looked at the b i n d i n g of A.cryptum to c h a l c o p y r i t e (CuFeS2) and other mineral ores t o determine whether s p e c i f i c adhesins mediate b i n d i n g . A modified ELISA b i n d i n g assay (the Ore ELISA) was developed to measure d i r e c t adherence. F i n e l y ground c h a l c o p y r i t e was bound i r r e v e r s i b l y to the w a l l s of an ELISA p l a t e , the organisms were added and a f t e r i n c u b a t i o n and washing, the number of attached b a c t e r i a were assessed by r e a c t i n g w i t h anti-A.crypturn antibody followed by goat a n t i - r a b b i t IgG conjugated t o a l k a l i n e phosphatase. This assay was found t o be s e n s i t i v e , r a p i d and r e p r o d u c i b l e . The Ore ELISA allowed d i r e c t b i n d i n g measurement i n the presence of v a r i o u s i n h i b i t o r s and provided a r a p i d screening method f o r adherence-defective mutants. Adherence was shown to be satu r a b l e and increased s l i g h t l y as pH decreased. A moderate increase i n b i n d i n g a f f i n i t y was recorded i n the presence of monovalent and d i v a l e n t c a t i o n s and EDTA. Various b a c t e r i c i d a l agents and pentose and hexose sugars had no e f f e c t on c h a l c o p y r i t e attachment. Reducing agents had l i t t l e e f f e c t on c e l l adherence. A strong increase i n i i adherence was observed i n the presence of surface a c t i v e agents. Bovine serum albumin and g e l a t i n were both found to markedly reduce mineral surface b i n d i n g . Competition f o r attachment s i t e s between A.cryptum and the aut o t r o p h i c a c i d o p h i l e , Thiobacillus ferrooxidans, showed that each organism binds to unique s i t e s on the c h a l c o p y r i t e surface. A.cryptum mutant s t r a i n s d i s p l a y i n g reduced adherence to c h a l c o p y r i t e were shown to l a c k a 31.6 kDa outer membrane p r o t e i n . i i i T A B L E O F C O N T E N T S I n t r o d u c t i o n 1 A c i d o p h i l e s 1 Adherence 19 M a t e r i a l s and Methods 37 B a c t e r i a 37 Media and stock maintenance 37 B u f f e r s 38 Bio-Rad adherence assay 38 Ore ELISA adherence assay 39 I s o l a t i o n of adherence v a r i a n t s 42 C e l l envelope p r o t e i n p r e p a r a t i o n 43 C e l l envelope s o l u b i l i z a t i o n 43 I n h i b i t i o n assay 44 C e l l m o d i f i c a t i o n 44 Competition assay 45 Hydrophobicity assay 45 Surface p r o t e i n p r e p a r a t i o n (SPP) 46 SDS-Polyacrylamide g e l e l e c t r o p h o r e s i s 46 Pre p a r a t i o n of IgG 47 Adsorption of IgG 47 Western b l o t 48 i v Immunogold bead l a b e l l i n g 4 9 E l e c t r o n microscopy 49 SPP i n h i b i t i o n assay 49 Antibody i n h i b i t i o n assay 50 Results 51 Bio-Rad assay f o r measuring A.cryptum adherence to c h a l c o p y r i t e 51 Development of the Ore ELISA assay 57 Adherence i n h i b i t i o n s t u d i e s 65 Competition stud i e s 78 I s o l a t i o n of adherence-defective v a r i a n t s 81 A n a l y s i s of adherence-defective mutants 85 L o c a l i z a t i o n of the 31.6 kDa p r o t e i n 87 D i s c u s s i o n 100 B i b l i o g r a p h y 109 v LIST OP TABLES v i LIST OF FIGURES Figure Page 1 Bio-Rad assay i l l u s t r a t i n g the adsorption of A.cryptum to c h a l c o p y r i t e 52 2 Bio-Rad assay demonstrating the r e l a t i o n s h i p between the number of c e l l s bound and the amount of Newmont added 53 3 Bio-Rad assay i l l u s t r a t i n g c e l l adsorption as a f u n c t i o n of adherence b u f f e r pH 54 4 Bio-Rad assay i l l u s t r a t i n g the r e l a t i o n s h i p between i n c u b a t i o n time and the number of A.cryptum c e l l s bound 55 5 Cr o s s - s e c t i o n of an Ore ELISA p l a t e 58 6 E f f e c t of assay pH on adsorption at 405 nm 59 7 Ore ELISA measuring the adsorption isotherm f o r A.cryptum b i n d i n g t o c h a l c o p y r i t e 66 8 Ore ELISA demonstrating the e f f e c t of adherence b u f f e r pH on A.cryptum b i n d i n g t o c h a l c o p y r i t e 67 9 R e l a t i o n s h i p between length of in c u b a t i o n and adherence as measured by the Ore ELISA assay 68 10 Adherence to d i f f e r e n t minerals 69 11 Ore ELISA measuring the bi n d i n g of T.ferrooxidans to c h a l c o p y r i t e 79 12 Competition between A.cryptum and T.ferrooxidans f o r b i n d i n g s i t e s on c h a l c o p y r i t e 80 13 Nitrosoguanidine s u r v i v a l curve 82 v i i 14 Western b l o t of SDS-PAGE c o n t a i n i n g p a r e n t a l and mutant whole c e l l l y s a t e s developed w i t h anti-A.cryptum antibody 86 15 SDS-PAGE of surface p r o t e i n preparations 88 16 Western b l o t of SDS-PAGE c o n t a i n i n g surface p r o t e i n preparations developed w i t h a n t i -A.cryptum antibody 89 17 Western b l o t of SDS-PAGE c o n t a i n i n g CEP preparations developed w i t h anti-31.6 kDa antibody 92 18 Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h anti-31.6 kDa antibody 94 19(a) Immunogold bead l a b e l l i n g of mutant-15 wi t h anti-31.6 kDa antibody 95 19(b) Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h non-immune serum 96 20 Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h anti-31.6 kDa antibody ( d i s r u p t i v e ) 97 21 Immunogold bead l a b e l l i n g of a p a r e n t a l s t r a i n A.cryptum c e l l p r i n t w i t h anti-31.6 kDa antibody 98 v i i i Dedicated to my w i f e , L e s l e y . i x ACKNOWLEDGEMENTS I t h a n k f u l l y acknowledge the support, encouragement and guidance of Dr. B. C. McBride. I a l s o g r a t e f u l l y acknowledge the f i n a n c i a l support of the U n i v e r s i t y of B r i t i s h Columbia and the Medical Research C o u n c i l of Canada. I extend my thanks to Drs. D. K i l b u r n and J . Smit f o r t h e i r guidance wh i l e s e r v i n g on my committee. I wish to thank Angela Joe, Drs. Daniel Grenier, Umadatt Singh and Nadarajah Ganeshkumar f o r s h a r ing t h e i r t e c h n i c a l e x p e r t i s e . I g r a t e f u l l y acknowledge Pauline Hannam f o r her as s i s t a n c e during some of the experiments i n c l u d e d i n t h i s document. I o f f e r my s i n c e r e thanks t o Laura W i l l i a m s , Dr. Helen Scott and Heather M e r i l e e s f o r t h e i r support and sense of humour. I would a l s o l i k e t o thank my f r i e n d s and fa m i l y f o r t h e i r encouragement, Andre Wong f o r h i s work w i t h the e l e c t r o n microscope and Bruce McCaughey and Harold Traeger f o r t h e i r photography. I extend s p e c i a l thanks to Lesley H e f f e l f i n g e r f o r a s s i s t i n g i n the pr e p a r a t i o n of t h i s manuscript. x INTRODUCTION Acidophiles A c i d o p h i l e s are unique among microorganisms i n that they l i v e at pHs below 5.0. A c i d o p h i l e s can be d i v i d e d i n t o two groups; f a c u l t a t i v e a c i d o p h i l e s and extreme a c i d o p h i l e s . F a c u l t a t i v e a c i d o p h i l e s , which are capable of growth at both n e u t r a l and a c i d i c pH, in c l u d e such b a c t e r i a l species as Streptococcus, Lactobacillus, and some species of Escherichia and Staphylococcus. Extreme a c i d o p h i l e s r e q u i r e an a c i d i c environment f o r growth and w i l l p e r i s h at n e u t r a l pH. Heterotrophic extreme a c i d o p h i l e s use a v a r i e t y of carbohydrates and organic acids as both an energy and carbon sources. Examples of h e t e r o t r o p h i c a c i d o p h i l e s are Acidiphilium spp. and Thiobacillus acidophilus. A u t o l i t h o t r o p h i c extreme a c i d o p h i l e s o b t a i n energy through the o x i d a t i o n of reduced i r o n and sulphur compounds and carbon through the f i x a t i o n of carbon d i o x i d e . Examples of aut o t r o p h i c a c i d o p h i l e s i n c l u d e Thiobacillus ferrooxidans and Thiobacillus thiooxidans, which o x i d i z e reduced s u l p h u r / i r o n and sulphur r e s p e c t i v e l y (Brock, 1979). The most common source of a c i d o p h i l i c microorganisms i s a c i d mine drainage (AMD) waters. Since t h e i r i s o l a t i o n and p o s t u l a t e d involvement i n AMD (Colmer, 1950), T h i o b a c i l l u s ferrooxidans has been the subject of considerable environmental research. AMD i s c h a r a c t e r i z e d by, a pH below 4.5, high f e r r i c i r o n and sulphate 1 l e v e l s , low concentrations of organic carbon, high l e v e l s of aluminum, calcium ions, s o l u b l e heavy metals (manganese and l e a d ) , and reduced l e v e l s of phosphate and n i t r o g e n (Johnson et a l . , 1979). There are two primary sources of mine waste: (1) T a i l i n g s , which are the by-products of mineral ore m i l l i n g and separation; (2) Overburden, which i s the s o i l and r e s i d u a l ore m a t e r i a l l e f t at the mine s i t e . Both forms of mine waste have s i g n i f i c a n t concentrations of reduced sulphur compounds which are o x i d i z e d through m i c r o b i a l a c t i o n to form AMD ( M i l l s , 1985). With the dwindling reserves of high grade ore and an i n c r e a s i n g demand f o r f o s s i l f u e l s , most fut u r e m e t a l - r e f i n i n g and f o s s i l f u e l production w i l l i n v o l v e surface s t r i p mining, r e s u l t i n g i n increased water p o l l u t i o n through mine drainage. The e f f e c t s of AMD on the environment are w e l l documented (Carpenter, 1925; Campbell and Lind , 1969; Yan, 1979; Johnson et a l . , 1978; N o r r i s et a l . , 1981; Olem, 1981). Most repo r t s have st u d i e d the physicochemical e f f e c t on the environment; the n a t u r a l f l o r a , plankton, v e g e t a t i o n , i n v e r t e b r a t e s and f i s h . The e f f e c t s of AMD on the m i c r o b i a l community has l a r g e l y been l i m i t e d to i r o n and sulphur o x i d i z i n g organisms such as T.ferrooxidans and T h i o b a c i l l u s thiooxidans ( B r i e r l e y , 1978). There i s a growing i n t e r e s t i n the e f f e c t s of AMD on h e t e r o t r o p h i c organisms and t h e i r c o n t r i b u t i o n , i f any, to the r e h a b i l i t a t i o n of a c i d mine p o l l u t e d water. In general most 2 h e t e r o t r o p h i c microbes are s e n s i t i v e to pH v a l u e below 5 or above 9 (Alexander , 1977) . In a d d i t i o n , meta ls such as Cu , C d , Pb and Zn, which are found i n h i g h l e v e l s i n AMD, are t o x i c t o many microorgani sms ( S t e r r i t t & L e s t e r , 1980; Zevenhuizen et a l . , 197 9) . Though c o n s i d e r a b l e r e s e a r c h has q u e s t i o n e d the e f f e c t s o f c e r t a i n a spec t s o f AMD on m i c r o o r g a n i s m s , c o m p a r a t i v e l y l i t t l e work has addres sed the m i c r o b i a l response and s u c c e s s i o n o f events f o l l o w i n g exposure t o AMD. Wassel e t a l . ( 1 9 8 3 ) s t u d i e d the water and sediment b a c t e r i a l communit ies f o r s t r e s s - r e l a t e d responses to a p o i n t source o f a c i d mine d r a i n a g e . In g e n e r a l , they found t h a t the d i v e r s i t y o f the m i c r o b i a l communit ies was s i g n i f i c a n t l y lower at the s i t e r e c e i v i n g AMD as compared to uncontaminated c o n t r o l s ; microorgan i sms at the contaminated s i t e s were more s i m i l a r t o each o t h e r than they were to the c o n t r o l s . M i l l s s u p p o r t e d these f i n d i n g s i n c o n c l u d i n g "that m i c r o b i a l communit ies d e v e l o p i n g i n extreme environments t e n d to be g e n e r a l i s t s , where as those i n mesic environments t e n d t o be s p e c i a l i s t s " ( M i l l s & M a l l o r y , 1987) . U n l i k e the a u t o t r o p h i c Thiobacillus s p p . , h e t e r o t r o p h i c b a c t e r i a were l o n g c o n s i d e r e d t r a n s i e n t s i n a c i d mine waters ( T u t t l e et a l . , 1968) . However, s p e c i a l l y adapted h e t e r o t r o p h i c organisms have been i s o l a t e d and are capab le o f growth i n AMD p o l l u t e d waters (Wichlacz e t a l . , 1981; Manning, 1975) . AMD i s not the o n l y source o f a c i d o p h i l i c h e t e r o t r o p h s , they have a l s o been i s o l a t e d from a c i d i c sewage and s o i l , and from what was presumed 3 t o be pure c u l t u r e s of T.ferrooxidans (Kishimoto, 1987; Har r i s o n , 1980; Zavarzin, 1972; Johnson & Kelso, 1983). Through DNA homology s t u d i e s , 25% of 23 s t a i n s of T.ferrooxidans were found to contain h e t e r o t r o p h i c contaminants (Harrison, 1982). One common contaminant was Thiobacillus acidophilus which i s described by Arkesteyn et a l . (1980) as a mixotrophic T h i o b a c i l l u s species. A second common contaminant contained a DNA G:C content (68-70%) beyond the range of the Thiobacillus species. H a r r i s o n et a l . (1981), proposed a new generic name, Acidiphilium gen. nov. (type species: Acidiphilium cryptum) and described i t as a m o t i l e , gram-negative, aerobic, mesophyllic, rod-shaped, h e t e r o t r o p h i c a c i d o p h i l e . This new a c i d o p h i l e was non-encapsulated, m o t i l e by a p o l a r or two l a t e r a l f l a g e l l u m and unable to o x i d i z e reduced sulphur or i r o n compounds. A.cryptum can u t i l i z e v a r i o u s carbohydrates as sol e carbon and energy sources. Operational pentose phosphate and Entner-Doudoroff pathways were i d e n t i f i e d by Shuttleworth et a l . (1985). Since i t s discovery i n 1981, four a d d i t i o n species of A c i d i p h i l i u m have been i s o l a t e d ; Acidiphilium angustum, Acidiphilium f a c i l i s , Acidiphilium rubrum (Wichlacz, 1986) and Acidiphilium organovorum (Lobos, 1986) . I t has been suggested that the main cause of v a r i a b l e o x i d a t i o n rates between d i f f e r e n t T. ferrooxidans c u l t u r e s ( K e l l y & Jones, 1978; Chang and Myerson, 1982; Roy et a l . , 1981; Gormely et a l . , 1975) i s a t t r i b u t e d t o h e t e r o t r o p h i c contaminants. H a r r i s o n et 4 a l . , suggested t h a t since A c i d i p h i l i u m and T h i o b a c i l l u s are so t e n a c i o u s l y a s s o c i a t e d i n c u l t u r e d i s o l a t e s , they may cross-feed i n the n a t u r a l environment (Harrison, 1980). The idea of m i c r o b i a l symbiosis between l i t h o t r o p h s and heterotrophs i s not new. I t i s commonly found, i n b i o l e a c h i n g operations, that mixed c u l t u r e s of b a c t e r i a have a higher mineral y i e l d than pure c u l t u r e of a u t o t r o p h i c organisms (Andrews et a l . , 1982; Detz, 197 9; Hoffman et a l . , 1981; Dugan, 1978; Andrews et a l . , 1987; N o r r i s & K e l l y , 1978). Many simple organic compounds have been found to i n h i b i t growth and i r o n and sulphur o x i d a t i o n by T. ferrooxidans ( T u t t l e & Dugan, 1976; Rao & Berger, 1970). Arkesteyn (1980) demonstrated that i n h i b i t i o n of i r o n - o x i d i z i n g T. ferrooxidans by c e r t a i n organic compounds (eg. 1.0 mM Glucose, ethanol, l a c t a t e , succinate, s e r i n e and aspartate) was r e l i e v e d when the mixotrophic T.acidophilus was added to the a u t o t r o p h i c T.ferrooxidans. A m u t u a l i s t i c r e l a t i o n s h i p i s b e l i e v e d t o occur between the i r o n / s u l p h u r o x i d i z i n g T.ferrooxidans and the s t r i c t l y sulphur o x i d i z i n g T.thiooxidans. As i r o n o x i d a t i o n proceeds, sulphur l a y e r s may coat the ore substrate and i n h i b i t f u r t h e r o x i d a t i o n . T.thiooxidans i s b e l i e v e d to o x i d i z e the accumulating sulphur and thus expose more ferrous substrate f o r T.ferrooxidans, however, general agreement on t h i s theory i s l a c k i n g . K e l l y et a l . i n 1978, described a symbiotic r e l a t i o n s h i p between the i r o n o x i d i z i n g a c i d o p h i l e , Leptospirillium ferrooxidans and the sulphur o x i d i z i n g Thiobacillus organoparus ( K e l l y et a l . , 1978). 5 The mechanisms by which organic compounds i n h i b i t T. ferrooxidans i s p o o r l y understood. Using e l e c t r o n micrographs, T u t t l e et a l . (1977), i l l u s t r a t e d c e l l envelope d i s r u p t i o n when T. ferrooxidans was exposed t o organic a c i d s . In 1970, Rao and Berger demonstrated passive p e r m e a b i l i t y to p y r u v i c a c i d at low pH, r e s u l t i n g i n a decreased i n t r a c e l l u l a r pH. Ingledew proposed that the t o x i c i t y of weak acids can be a t t r i b u t e d to t h e i r accumulation i n the c e l l matrix i n response to the pH d i f f e r e n c e between the cytoplasm and the supporting medium (Ingledew, 1982). The a b i l i t y of weak acids to permeate membranes req u i r e s that the a c i d be protonated; t h i s , i n t u r n , w i l l l e a d to a c i d i f i c a t i o n of the c e l l m atrix. Alexander et a l . , supported t h i s p r e d i c t i o n by demonstrating t h a t organic a c i d t o x i c i t y i s d i r e c t l y r e l a t e d to the pKa values of each a c i d (Alexander et a l . , 1987). The source of the i n h i b i t o r y organic compounds was i n v e s t i g a t e d by Schnaitman and Lundgren i n 1965. Using r a d i o i s o t o p e s , i t was shown that the m a j o r i t y of the p y r u v i c a c i d accumulating i n spent media was from C0 2 f i x e d by T. ferrooxidans and subsequently rele a s e d i n t o the media (Schnaitman & Lundgren, 1965). From these observations, the g e n e r a l l y accepted c o n c l u s i o n i s that end product i n h i b i t i o n plays an important r o l e i n auto t r o p h i c a c i d i p h i l i c growth on mineral s u b s t r a t e s . Contrary to the de t r i m e n t a l e f f e c t of a c i d mine drainage, the 6 b e n e f i c i a l uses of a c i d o p h i l e s have been p r a c t i c e d f o r thousands of years. B i o l e a c h i n g , through the o x i d a t i o n of metal sulphides, has been s u c c e s s f u l l y a p p l i e d on an i n d u s t r i a l s c a l e to the recovery of copper and uranium from low grade ores and t a i l i n g s . I t has been reported that 15-20% of commercially produced copper i n the U.S. i s achieved through b i o l e a c h i n g w i t h a d e f i n i t e p o t e n t i a l t o expand to other ores and ore concentrates (Agate & Khinvasara, 1985). Over the l a s t three decades, s e v e r a l a r t i c l e s have been w r i t t e n on b i o l e a c h i n g . B i o l e a c h i n g i n v o l v e s the b a c t e r i a l o x i d a t i o n of i r o n , elemental sulphur and mineral sulphides such as p y r i t e (FeS2) . This process r e s u l t s i n d i r e c t and i n d i r e c t s o l u b i l i z a t i o n of metals t o be commercially e x t r a c t e d . The d i r e c t mechanism i n v o l v e s the d i r e c t b i o l o g i c a l o x i d a t i o n of an i n s o l u b l e d i v a l e n t metal sulphide t o s o l u b l e metal and sulphate. I t g e n e r a l l y b e l i e v e d that d i r e c t attack r e q u i r e s b a c t e r i a l attachment to the mineral since the substrate i s i n s o l u b l e (Bennett & T r i b u t s c h , 1978; Berry & Murr, 1978; Tr i b u t s h , 197 6). Reactions 1 and 2 describe the b a c t e r i a l o x i d a t i o n of elemental sulphur and p y r i t e by the d i r e c t mechanism. (1) 2 S° + 3 0 2 + 2 H20 - — 2 H2S04 (2) 4 FeS 2 + 15 0 2 + 2 H20 2 Fe 2(S0 4) 3 + 2 H2S04 Any b i v a l e n t metal can be s u b s t i t u t e d f o r ferrous sulphide (FeS2) . The i n d i r e c t mechanism of m i c r o b i a l l e a c h i n g i n v o l v e s three steps. (a) In the a c i d environment created by m i c r o b i a l l e a c h i n g , ferrous sulphate i s produced by the chemical o x i d a t i o n of p y r i t e (FeS2) ( r e a c t i o n 3) . (b) B a c t e r i a l o x i d a t i o n of ferrous i r o n to f e r r i c i r o n ( r e a c t i o n 4) (Lacey & Lawson, 1970). (c) Chemical reduction of f e r r i c sulphate through the o x i d a t i o n of a metal sulphide to regenerate ferrous sulphate ( r e a c t i o n 5) (Singer & Stumm, 1970) . (3) 2 FeS 2 + 7 0 2 + 2 H20 2 FeS0 4 + 2 H2S04 (4) 4 FeS0 4 + 0 2 + 2 H2S04 —*~ 2 Fe 2(S0 4) 3 + 2 H20 (5) Fe 3 + + MS Fe 2 + + M2+ + S° The metal sulphide (MS) o x i d i z e d t o a s o l u b l e i o n i c form i n r e a c t i o n 5 f a c i l i t a t e s the establishment of the f e r r o u s - f e r r i c i r o n c y c l e which i s c h a r a c t e r i s t i c of the i n d i r e c t mechanism. B a c t e r i a l o x i d a t i o n of ferrous i r o n t o r e p l e n i s h f e r r i c i r o n l e v e l s ( r e a c t i o n 4) i s c r i t i c a l f o r the maintenance of t h i s c y c l e . I f r e a c t i o n 5 i s not allowed to occur, then f e r r i c sulphate w i l l r e a d i l y react w i t h water t o form f e r r i c hydroxide and s u l p h u r i c a c i d . Since a l l re a c t i o n s i n the i n d i r e c t mechanism occur i n s o l u t i o n , attachment i s not considered necessary. Though separate systems f o r i r o n and sulphur o x i d a t i o n are e v i d e n t (Beck & Brown, 1968), they appear to f u n c t i o n c o n c u r r e n t l y ( S i l v e r m a n , 1967) . S u l p h u r i c a c i d i s an end p r o d u c t f o r b o t h r e a c t i o n sys tems. The r e s u l t a n t low pH p l a y s an impor tant r o l e i n b i o l e a c h i n g . A c i d i c pH p r o v i d e s s e l e c t i v e p r e s s u r e f o r the enr ichment o f a c i d o p h i l i c l i t h o t r o p h s and h e t e r o t r o p h s . At a low pH, f e r r o u s i r o n i s s t a b l e and w i l l not a u t o - o x i d i z e t o f e r r i c i r o n , t h e r e f o r e , t h i s impor tant s u b s t r a t e remains a v a i l a b l e to the b a c t e r i a . Under low pH c o n d i t i o n s , a membrane p o t e n t i a l o f a p p r o x i m a t e l y -10 mV between the a c i d i c environment and the n e u t r a l i n t e r n a l pH o f T.ferrooxidans i s g e n e r a t e d (Ingledew & C o b l e y , 1980). T h i s c r e a t e s an e l e c t r o c h e m i c a l p r o t o n a c t i v i t y d i f f e r e n c e o f 256 mV which i s s u f f i c i e n t f o r ATP g e n e r a t i o n , as demonstrated by Ingledew et a l . V a r i o u s parameters e f f e c t b i o l e a c h i n g r a t e s (Lundgren & S i l v e r , 1980) . I n c r e a s i n g t e m p e r a t u r e , w i t h i n a range ( 2 5 - 4 5 ° C ) , r e s u l t s i n not o n l y an i n c r e a s e i n c h e m i c a l r e a c t i o n r a t e s , but a l s o an i n c r e a s e i n b a c t e r i a l metabo l i sm. The pH o f 1 .0 -2 .5 i s c o n s i d e r e d o p t i m a l f o r f e r r o u s i r o n o x i d a t i o n . As w i t h most m i c r o o r g a n i s m s , a c i d o p h i l e s r e q u i r e s t a n d a r d m i n e r a l s a l t s . In many c a s e s , depending on the ore c o m p o s i t i o n , i t may be o n l y n e c e s s a r y t o supplement the l e a c h i n g media w i t h ammonia and phosphate . C0 2 and 0 2 a v a i l a b i l i t y i s d i r e c t l y r e l a t e d t o the l e a c h i n g e f f i c i e n c y . Both gases may become l i m i t i n g i f p u l p 9 d e n s i t i e s are too high. The p a r t i c l e s i z e i s a l s o an important parameter; decreased p a r t i c l e s i z e causes an increase i n surface area r e s u l t i n g i n an increased l e a c h i n g e f f i c i e n c y (Torma, 1977). Torma and Bosecker addressed the growing i n t e r e s t i n commercially a p p l i e d bio-hydrometallurgy by p r o v i d i n g a systematic review of current techniques i n mineral l e a c h i n g by microorganisms. Various methods of i n d u s t r i a l b i o l e a c h i n g have been developed; a i r l i f t p e r c o l a t o r s , column le a c h i n g , s t a t i o n a r y l e a c h i n g and a g i t a t e d vat l e a c h i n g . They found that f o r best r e s u l t s , the r a t e of oxygen and carbon d i o x i d e mass t r a n s f e r must be at a maximum. The authors s t r e s s that i n order f o r commercial a p p l i c a b i l i t y , an increased understanding of the m i c r o b i a l leach process and the symbiotic growth phenomena i s e s s e n t i a l (Torma & Bosecker, 1982) . One a p p l i c a t i o n of i n d u s t r i a l b i o l e a c h i n g which i s of growing i n t e r e s t i s the d e s u l p h u r i z a t i o n of c o a l as a means of curbing sulphur d i o x i d e and n i t r o g e n oxide emissions. Both emissions i n t e r a c t w i t h water molecules i n the atmosphere to form s u l p h u r i c a c i d and n i t r i c a c i d , hence a c i d r a i n ( L a B a s t i l l e , 1981). P u b l i c concerns of a c i d r a i n has encouraged implementation of the Clean A i r Act i n the U.S. which r e s t r i c t s the sulphur content of combustible c o a l t o 0.7%. Unfortunately, h a l f of the U.S. c o a l reserves i s bituminous i n nature w i t h i t s m a j o r i t y c o n t a i n i n g greater than 1% sulphur ( M i l l s , 1985). This problem i s compounded by the f a c t that 47% of the U.S. energy needs r e s u l t from c o a l combustion and a p r e d i c t e d 5% annual increase i n domestic energy needs i s expected f o r the next s e v e r a l decades (Detz, 1979) . Two common concerns among researchers designing d e s u l p h u r i z a t i o n treatments are economic f e a s i b i l i t y and reduced residence time. Two n o n - b i o l o g i c a l approaches are f l u e gas d e s u l p h u r i z a t i o n and p r e s s u r i z e d f l u i d i z e d bed combustion, however, high costs and excess waste have l i m i t e d t h e i r usefulness (Detz, 1979). The suppression of p y r i t i c sulphur f l o a t a t i o n during c o a l c l e a n i n g i s another approach. Boateng and P h i l l i p s found that by t r e a t i n g the c o a l w i t h kerosene and lime, you encourage c o a l f l o a t a t i o n and discourage p y r i t e f l o a t a t i o n (Boateng & P h i l l i p s , 1977). Therefore, the sulphur c o n t a i n i n g p y r i t e i s removed from the c o a l p r i o r t o combustion, v a r i a t i o n s to t h i s process were attempted by Kempton e l a l . i n 1980 and Townsley et a l . i n 1987. Both researchers showed th a t by c o n d i t i o n i n g the c o a l w i t h T.ferrooxidans, the n a t u r a l f l o a t a b i l i t y of p y r i t e was s i g n i f i c a n t l y reduced. M i c r o b i a l d e s u l p h u r i z a t i o n through b i o l e a c h i n g i s considered the l e a s t expensive route t o precombustion removal of p y r i t e from a broad range of coals (Detz, 1979; Dugan, 1986). Huber et a l . (1984) provided an a n a l y s i s of m i c r o b i a l d e s u l p h u r i z a t i o n of c o a l . Using a mixed flow r e a c t i o n followed by a plug flow r e a c t o r c o n f i g u r a t i o n , o v er 90% o f t h e p y r i t e was removed at an o v e r a l l 9 day r e s i d e n c e t i m e . The k i n e t i c s o f c o a l d e s u l p h u r i z a t i o n was r e c e n t l y r e v i e w e d by Andrews e t a l . (1988). C r i t i c a l 0 2 a v a i l a b i l i t y was met t h r o u g h a i r s p a r g i n g o f s l u r r i e s up t o 50% (Wt/Wt) c o n t a i n i n g 1% s u l p h u r . Workers found t h a t by r e c y c l i n g c e l l c u l t u r e s , a p o p u l a t i o n o f h e t e r o t r o p h s e s t a b l i s h e d t h e m s e l v e s and i n c r e a s e d p r o c e s s r a t e s not o n l y by removing T.ferrooxidans i n h i b i t o r s but a l s o by i n c r e a s i n g C0 2 l e v e l s such t h a t an exogenous so u r c e o f C0 2 was not n e c e s s a r y . Q u e s t i o n s w h i c h have r e s u l t e d i n h e a t e d debate among r e s e a r c h e r s i n t h i s f i e l d a r e : I s attachment n e c e s s a r y f o r m i n e r a l l e a c h i n g , and i f so, i s t h e adherence s e l e c t i v e and by what mechanism does i t o c c u r ? C l e a r l y , t h e r e i s an advantage t o t h e organism t o be a t t a c h e d t o t h e m i n e r a l s u r f a c e , s i n c e i t i s t h e n p r o x i m a l t o t h e i n s o l u b l e o x i d i z a b l e s u b s t r a t e . E a r l y e v i d e n c e o f T.ferrooxidans d i r e c t i n t e r a c t i o n w i t h s u l p h u r was p r o v i d e d by Voger (Voger & Umbreit, 1941). S c h a e f f e r d e m onstrated t h a t T.thiooxidans t e n a c i o u s l y b i n d s t o s u l p h u r and c o n c l u d e d t h a t attachment i s a p r e r e q u i s i t e f o r s u l p h u r o x i d a t i o n ( S c h a e f f e r & H o l b e r t , 1963). The s t r e n g t h and i r r e v e r s i b i l i t y o f m i n e r a l s u r f a c e b i n d i n g was f u r t h e r demonstrated by B a l d e n s p e r g e r e t a l . i n 1974 w i t h T. thiooxidans and T. denitrificans and by Myerson and K l i n e i n 1983 w i t h T.ferrooxidans. I n c r e a s i n g pH appeared t o have no e f f e c t on T.ferrooxidans d e s o r p t i o n nor d i d repeat washing (Tuovinen et a l . , 1983). M i l d s o n i c a t i o n or treatment w i t h s u r f a c t a n t s were a l s o i n e f f e c t i v e at removing T.ferrooxidans c e l l s bound to c h a l c o p y r i t e (McGorman et a l . , 1969) . T.ferrooxidans appears t o bi n d e x t e n s i v e l y t o mineral surfaces. Wakao et a l . i n 1984 recorded t h a t almost 100% of a T.ferrooxidans c e l l suspension adsorbed to e i t h e r the p y r i t e c r y s t a l s or the w a l l s of the gl a s s tube. McGorman found that over 97% of a c e l l suspension bound to c h a l c o p y r i t e (McGorman et a l . , 1969). Gormely and Duncan reported that 65% of a T.ferrooxidans p o p u l a t i o n a s s o c i a t e d d i r e c t l y w i t h the z i n c sulphide substrate (Gormely & Duncan, 1974). The adherence to mineral surfaces occurs q u i t e r a p i d l y . Myerson and K l i n e observed t h a t over h a l f the c e l l s i n a suspension adsorbed to coa l p a r t i c l e s or glass beads w i t h i n f i v e minutes (Myerson & K l i n e , 1983). Twenty-five percent of a T.ferrooxidans c e l l suspension absorbed w i t h i n f i v e minutes to e i t h e r p y r i t e p a r t i c l e s , f l u o r - a p a t i t e or gl a s s beads (Tuovinen et a l . , 1983). Very s i m i l a r r e s u l t s were recorded by D i S p i r i t o et a l . i n 1983. U n t i l r e c e n t l y , the m a j o r i t y of the evidence demonstrating d i r e c t i n t e r a c t i o n between microorganisms and mineral surfaces has been from e l e c t r o n microscope stud i e s (Lundgren & Tano, 1978; Tr i b u t s c h , 1976; Wakao et a l . , 1984; Bennett & T r i b u t s c h , 1978; Gromely et a l . , 1974; Murr & Berry, 1976; Wiess, 1973; Duncan & Drummond, 1973; H i l t u n e n , 1981; K e l l e r & Murr, 1982; B r i e r l e y e t a l . , 1973). S c a n n i n g e l e c t r o n m i c r o s c o p e (SEM) s t u d i e s ( B e r r y & Murr, 1978; K e l l y e t a l . , 1979) suggest t h a t T.ferrooxidans attachment o c c u r s s e l e c t i v e l y t o s u l p h i d e phases r a t h e r t h a n t o the s i l i c a t e m a t r i x o f p r e p a r e d m i n e r a l samples. Bennett & T r i b u t s c h p e r f o r m e d SEM s t u d i e s on p y r i t e c r y s t a l s w hich were p l a c e d i n a T.ferrooxidans c u l t u r e f o r 2 y e a r s . They obse r v e d e t c h e d p i t s i n t h e shape o f b a c t e r i a l c e l l s w hich were o r i e n t e d i n a " p e a r l - l i k e " s t r i n g . These l i n e s o f b a c t e r i a l c o r r o s i o n were o f t e n i n t e r s e c t e d a t r i g h t a n g l e s by o t h e r c o r r o s i o n l i n e s , t h e r e f o r e r e s e m b l i n g th e c u b i c s t r u c t u r e o f c r y s t a l l i n e p y r i t e . B a g d i g i a n and Myerson (198 6) demonstrated t h a t T.ferrooxidans s e l e c t i v e l y a dsorb t o exposed p y r i t e c r y s t a l s d i s p e r s e d t h r o u g h o u t t h e o r g a n i c c o a l m a t r i x . F u r t h e r m o r e , p r e f e r e n t i a l attachment was obs e r v e d a l o n g f r a c t u r e l i n e s and d i s l o c a t i o n p o i n t s o f t h e p y r i t e c r y s t a l . S e l e c t i v e a d s o r p t i o n t o p y r i t e c r y s t a l s was a l s o d emonstrated by Kempton e t a l . (1980) and Townsley e t a l . (1987) as a means o f d e s u l p h u r i z a t i o n o f c o a l ( p r e v i o u s l y d i s c u s s e d ) . A c c o r d i n g t o Weertman and Weertman (1964), s t r e s s and s t r a i n e n e r g i e s a r e s t o r e d i n d i s l o c a t i o n s i t e s i n t h e form o f c r y s t a l f o r m a t i o n s and l a t t i c e v a c a n c i e s . W i t h t h i s i n mind, Andrews found t h a t d i f f u s i o n o f s u l p h u r t o d i s l o c a t i o n s i t e s o f p y r i t e c r y s t a l s was s i g n i f i c a n t and as such, i t would be advantageous f o r t h e b a c t e r i a t o adsorb t o t h e e n e r g e t i c a l l y r i c h s u l p h u r w h i c h accumulates a t th e d i s l o c a t i o n s i t e (Andrews et a l . , 1988) . S t u d i e s i n v o l v i n g s u r f a c t a n t s and w e t t i n g agents have a l s o produced ev idence l i n k i n g c e l l - s u r f a c e i n t e r a c t i o n w i t h i n c r e a s e d r a t e s o f i r o n and s u l p h u r o x i d a t i o n . I t i s l i k e l y t h a t these substances e f f e c t the i n t e r a c t i o n o f b a c t e r i a and the m i n e r a l s u r f a c e . A c c o r d i n g t o Kingma et a l . (1979), Tween reduces the energy r e q u i r e d by the b a c t e r i u m t o overcome the dynamic s u r f a c e t e n s i o n between the s u b s t r a t e and the l i q u i d medium, thus f a c i l i t a t i n g c o n t a c t between the b a c t e r i u m and the s u b s t r a t u m . S t a r k e y et a l . observed an i n c r e a s e d s u l p h u r o x i d a t i o n i n shake f l a s k c u l t u r e s when F e r g i t o l - 0 8 or Tween 80 was added (S tarkey , 1956) . An enhanced r a t e and ex tent o f l e a c h i n g was r e c o r d e d by Duncan et a l . (1964) when e i t h e r Tween, 20, 40, 60 or 80, or T r i t o n X-100 was added t o T. ferrooxidans c u l t u r e s growing on c h a l c o p y r i t e . Moreover , they found t h a t the Tween t o ore r a t i o was more i m p o r t a n t , w i t h r e g a r d s to l e a c h i n g r a t e s , than the Tween t o media r a t i o , s u g g e s t i n g t h a t Tween a c t s to i n c r e a s e the c o n t a c t between the m i n e r a l s u r f a c e and the m i c r o o r g a n i s m s . A s i d e from i n c r e a s i n g s e l e c t i v e c o n t a c t between the b a c t e r i u m and the subs tra tum (Duncan et a l . , 1973), Tween was found to i n c r e a s e the r a t e o f c o n t a c t by e l i m i n a t i n g the l a g p e r i o d p r i o r to p y r i t e o x i d a t i o n (Roy et a l . , 1981) . Torma et a l . (1976), r e f u t e d these f i n d i n g by r e c o r d i n g a reduced c h a l c o p y r i t e o x i d a t i o n by T.ferrooxidans i n the presence o f s u r f a c e a c t i v e agents (Tween 20, 40, 60 and 80) and o r g a n i c s o l v e n t s . They e x p l a i n e d t h a t the surface a c t i v e agents reduced the surface t e n s i o n and oxygen concent r a t i o n of the medium r e s u l t i n g i n a decreased o x i d a t i o n r a t e . There i s evidence suggesting t h a t surface a c t i v e agents may i n f a c t be endogenously produced w i t h i n b i o l e a c h i n g systems. Wakao et a l . (1983), proposed that the enhanced p y r i t e o x i d a t i o n observed i n T.thiooxidans and T. ferrooxidans mixed c u l t u r e s was due to a s u r f a c t a n t , i d e n t i f i e d as p h o s p h o t i d y l i n o s i t o l (Schaeffer & Umbreit, 1963) secreted by T.thiooxidans. This s u r f a c t a n t i s b e l i e v e d t o reduce the T.ferrooxidans adherence, t h e r e f o r e a l l o w i n g i t to o x i d i z e i r o n more r e a d i l y i n suspension. However, i n 1961, Jones and Starkey i d e n t i f i e d a s u r f a c t a n t produced by T.thiooxidans which enhanced sulphur o x i d a t i o n by wetting the substrate and i n c r e a s i n g b a c t e r i a l adherence. Evidence r e l a t i n g the importance of attachment and substrate o x i d a t i o n was r e c e n t l y reviewed by Espejo et a l . (1987) and Yeh et a l . (1987). T.ferrooxidans growth on sulphur p r i l l s was found to be s e l e c t i v e and s a t u r a b l e . Radioisotope stud i e s had shown that only the attached c e l l s were a c t i v e l y d i v i d i n g . The unattached c e l l s l o s t v i a b i l i t y w i t h a h a l f - l i f e of 3.5 days (Espejo et a l . , 1987). Yeh et a l . (1987) used e p i f luorescence microscopy t o measure mineral adherence and metabolic a c t i v i t y . Through d i f f e r e n t i a l fluorescence, they concluded that the c e l l s most a c t i v e l y i n v o l v e d i n the l e a c h i n g process are those attached to the mineral surface. In general, attachment during b a c t e r i a l l e a c h i n g i s advantageous f o r the microorganism. In a d d i t i o n to clo s e p r o x i m i t y of subst r a t e , attached organisms show greater r e s i s t a n c e t o pH changes, temperature changes and end product t o x i c i t y (Karamanev & Nikolov, 1988). There have been few complete stud i e s on the adherence k i n e t i c s of a c i d o p h i l e s . Adsorption to mineral surfaces appears to occur independently of pH, u n l i k e adsorption to glass beads which increased w i t h decreasing pH (Takakuwa et a l . , 197 9; Tuovinen et a l . , 1983; D i S p i r i t o et a l . , 1983). C e l l v i a b i l i t y was not a requirement f o r adherence (Beck, 1967), however, Tuovinen et a l . observed a s l i g h t o v e r a l l r eduction i n adherence w i t h u l t r a v i o l e t l i g h t t r e a t e d c e l l s . Adsorption experiments by Badigian et a l . (1986) suggests t h a t adsorption occurs through a combination of f i r s t order, r e v e r s i b l e and second order i r r e v e r s i b l e k i n e t i c s . Another a c i d o p h i l e of i n t e r e s t concerning mineral surface attachment i s the t h e r m o p h i l i c l i t h o t r o p h , Sulfolobus. Weiss (1973), through SEM s t u d i e s , observed Sulfolobus adhering t o sulphur p a r t i c l e s v i a a p i l u s and c o r r e l a t e d attachment w i t h the number and lengths of the surface p i l i . By in c u b a t i n g Sulfolobus i n sulphur, S h i w e r s et a l . (1973) saw a gradual increase i n the number of attached organisms. They a l s o c o r r e l a t e d increased sulphur o x i d a t i o n w i t h increased attachment. A comprehensive study of Sulfolobus attachment to c h a l c o p y r i t e drew the f o l l o w i n g conclusions (Berry & Murr, 1976): 1) . Strong support that d i r e c t contact i s necessary for chalcopyr i te ox idat ion . 2) . Evidence of p r e f e r e n t i a l attachment by Sulfolobus to mineral surfaces . 3) . Attachment corre la ted with i ron and copper d i s s o l u t i o n . 4) . Increased surface area corresponds with an increase i n c e l l attachment. 5) . Evidence of "biomatter" production at b a c t e r i a l attachment s i t e s . The molecular mechanism by which acidophi les attach to mineral surfaces i s poorly understood. Takakuwa et a l . (1979) postulated the involvement of sul fhydryl-groups i n T.thiooxidans adherence to sulphur p a r t i c l e s since su l fhydry l -b ind ing reagents blocked attachment. This view i s disputed by Bryant et a l . (1984) who i d e n t i f i e d a glycocalyx-mediated adsorption of T.albertis to sulphur d i s c s . LPS extract ion caused reduced adherence to p y r i t e , sulphur or f l o u r o - a p a t i t e , though, i t i s l i k e l y that other surface components would also be removed during the extract ion (DeSpirito et a l . , 1983). In addi t ion to f l a g e l l a , surface appendages have been reported to ex is t on Thiobacillus spp. (Gonzalez et a l . , 1987; D i S p i r i t o et a l . , 1981), however, the function of the appendages has not been determined. 18 Adherence M a r s h a l l et a l . (1980) d i v i d e s adherence i n t o three separate c a t e g o r i e s : A) S p e c i f i c permanent adhesion. This may a l s o be c a l l e d a c t i v e adherence i n which the b a c t e r i a attaches to a surface by means of complementary surface s t r u c t u r e s . The m a j o r i t y of t h i s form of adhesion has been described i n b a c t e r i a - e p i t h e l i u m i n t e r a c t i o n s . B) N o n - s p e c i f i c permanent adhesion, i s found i n microorganisms which adhere to a v a r i e t y of surfaces i n t h e i r n a t u r a l environment. The surfaces o f t e n d i f f e r c o n s i d e r a b l y suggesting that a n o n - s p e c i f i c mechanism such as polymer-bridging may be i n v o l v e d . C) Temporary adhesion, i s observed i n g l i d i n g b a c t e r i a i n which contact to a s o l i d surface i s necessary f o r movement. Adherence i s a very complex process and has been the subject of numerous s t u d i e s . To understand the mechanism of adherence, the researcher must consider charge, w e t t a b i l i t y , and adsorbed medium components ( e l e c t r o l y t e s and p r o t e i n s ) . Moreover, the observer must not assume th a t the c e l l surface physiology of the b a c t e r i a remains s t a t i c throughout the adherence r e a c t i o n ; i n t e r a c t i o n w i t h an i n t e r f a c e can induce changes i n b a c t e r i a l surfaces (Fl e t c h e r et a l . , 1980). Adhesion has been described to proceed from a r e v e r s i b l e s t a t e i n v o l v i n g n o n - s p e c i f i c bonding to an i r r e v e r s i b l e permanent i n t e r a c t i o n (Marshall et a l . , 1971). Doyle et a l . (1982) p o s t u l a t e d that permanent i r r e v e r s i b l e adherence i s achieved through the a d d i t i v e e f f e c t of n o n - s p e c i f i c i n t e r a c t i o n s a f t e r i n i t i a l contact. The complexity of b a c t e r i a l adherence i s e x e m p l i f i e d by Doyle's i n t e r p r e t a t i o n of p o s i t i v e c o o p e r a t i v i t y i n Streptococcus sanguis b i n d i n g to h y d r o x y l a p a t i t e (Nesbitt et a l . , 1982), i n which the adsorption of one c e l l encouraged the b i n d i n g of adjacent c e l l s . He suggests that once a c e l l binds, p e l l i c l e p r o t e i n s adjacent may change conformation c r e a t i n g new receptors and encouraging the adherence of other c e l l s . The molecular mechanism of adherence has been d i v i d e d i n t o four forms: (1) Chemical bonds which in c l u d e e l e c t r o s t a t i c , covalent and hydrogen bonds (Marshall, 1980). (2) Dipole i n t e r a c t i o n s such as d i p o l e - d i p o l e , dipole-induced d i p o l e and i o n - d i p o l e i n t e r a c t i o n s (Heckels et a l . , 1976; Stotzky, 1980). (3) Hydrophobic bonding (Doyle et a l . , 1982; Beachey, 1981). (4) L e c t i n - l i k e bonding (Duguid et a l . , 1966; C i s a r et a l . , 1983). 20 The f i r s t three types of adherence are g e n e r a l i z e d as being non-s p e c i f i c i n nature. Whereas l e c t i n - l i k e bonding i s a h i g h l y s p e c i f i c form of adherence. Chemical bonding, d i p o l e - i n t e r a c t i o n s and hydrophobic bonding are considered physicochemical adherence mechanisms and are discussed below. Using w e l l - d e f i n e d , n o n - l i v i n g systems, Rutte r & Vincent (1984), attempted to r e l a t e s t u d i e s of p a r t i c l e adhesion t o the understanding of m i c r o b i a l adherence to surfaces. They recognized a l i m i t a t i o n of such an approach was that surface charges are not s t a t i c and are often adaptive. A t r u e physicochemical e q u i l i b r i u m may not be reached because m i c r o b i a l attachment does not occur i n a c l o s e d system; i n t e r n a l or surface s t r u c t u r a l changes to the microorganism may occur during the adherence process. They s t r e s s that i n order t o understand the adhesive j u n c t i o n between a c e l l and substratum, a l l i n t e r a c t i o n s i n v o l v i n g the surfaces and the environment must be considered. In order f o r surfaces of s i m i l a r charge to come i n t o contact w i t h each other, a l a r g e free energy b a r r i e r must be overcome. They define t h i s free energy based on van der Waals forces and the overlap of the e l e c t r i c a l double l a y e r a s s o c i a t e d w i t h charged groups on each surface. This energy b a r r i e r i s i n f l u e n c e d by the e l e c t r o l y t e c o n c e n t r a t i o n i n the surrounding medium. As the e l e c t r o l y t e c oncentration i n c r e a s e s , the energy b a r r i e r decreases u n t i l a net a t t r a c t i o n occurs between surfaces. In cases i n v o l v i n g surfaces of opposite charge, the net e f f e c t i s a strong a t t r a c t i o n which i s only m i l d l y decreased w i t h increased e l e c t r o l y t e c o n c e n t r a t i o n . The p o i n t s discussed i n the preceding paragraph address long-range forces necessary to b r i n g two surfaces i n t o c l o s e p r o x i m i t y . However, short-range f o r c e s , i n the form of d i p o l e i n t e r a c t i o n s and hydrogen bonding, are necessary to achieve adhesion between the two surfaces. In the case of two h y d r o p h i l i c surfaces, there i s a net increase i n energy r e q u i r e d to d i s p l a c e the water molecules, r e s u l t i n g i n short-range r e p u l s i o n . The opposite e f f e c t occurs when two hydrophobic surfaces i n t e r a c t due t o a net decrease i n energy r e q u i r e d f o r water displacement. Macromolecules which l a y e r the surfaces of micro-organisms, can e f f e c t m i c r o b i a l adherence: Macromolecules, shed i n t o the environment, can le a d to increased s o l u t i o n v i s c o s i t y and polymer bridge formation. The l a t t e r occurs when the macromolecule co-adsorbs onto two separate surfaces. Bridge formation i s oft e n enhanced by d i v a l e n t c a t i o n s which l i n k polymers through a c i d i c groups. As more p a r t i c l e s of s i m i l a r surface charge adsorb t o a surface, there are l a t e r a l i n t e r a c t i o n s which must be considered. The 22 nature of these forces are dependant on the e l e c t r o l y t e c o n c e n t r a t i o n : In low e l e c t r o l y t e concentrations, adsorbing p a r t i c l e s tend to repulse one another. However, under high e l e c t r o l y t e concentrations, the l a t e r a l forces are a t t r a c t i v e . At intermediate e l e c t r o l y t e concentrations a compromise between a t t r a c t i v e and r e p u l s i v e forces i s reached. This i s d e f i n e d as the second minimum i n which p a r t i c l e s are maintained at some distance from the substratum surface by the opposing f o r c e s . Second minimum adherence i s of low a f f i n i t y and r e v e r s i b l e . High a f f i n i t y , i r r e v e r s i b l e adherence i s achieved only i f the necessary f r e e energy to overcome the primary and l a t e r a l forces i n a v a i l a b l e . Rutter and Vincent summarize by acknowledging that long-range and short-range forces w i l l not completely d i c t a t e a p a r t i c l e s adhesive behaviour. These forces f u n c t i o n t o r e v e r s i b l y h o l d a p a r t i c l e adjacent t o a surface, t h e r e f o r e a l l o w i n g the opportunity f o r i r r e v e r s i b l e adsorption. The p a r t i c l e must withstand hydrodynamic and shear forces from bulk medium flow. U l t i m a t e l y , the p a r t i c l e / s u r f a c e s p e c i f i c i t i e s w i l l draw the f i n a l l i n k between a c e l l and the substratum. By studying polymer adsorption t o surfaces, Robb et a l . (1984) p r e d i c t s the i n f l u e n c e of e x o c e l l u l a r polymers on b a c t e r i a adherence to surfaces. Polymer adsorption to surfaces appears to be i r r e v e r s i b l e . This i s p r i m a r i l y due to the formation of a 23 l a r g e number of r e l a t i v e l y weak bonds between the polymer and the substratum. Negatively charged b a c t e r i a adhering v i a polymers t o ne g a t i v e l y charged surface, i l l u s t r a t e s the streng t h of polymer adsorption. Polymer a c t i v e s i t e s r e s p o n s i b l e f o r adsorption i n c l u d e p o s i t i v e and n e g a t i v e l y charged regions of e x o c e l l u l a r polysaccharides and p r o t e i n s . Since polymers are often h i g h l y charged, the i o n i c s trength of the media i s an important f a c t o r concerning polymer-surface adsorption. The a f f i n i t y of a c e l l surface polymers f o r the solvent i s important i n preventing the c e l l from aggregating i n the s o l u t i o n and maintaining the polymer i n an extended s t a t e , thus, a v a i l a b l e f o r substratum i n t e r a c t i o n . Polymer extension from the c e l l surface i s oft e n s u f f i c i e n t enough to contain the net negative charge of the c e l l w a l l . As a r e s u l t , the net charge of the c e l l i s a t t r i b u t e d to the polymer i t s e l f . K j e l l e b e r g (1984), through the study of var i o u s environmental f a c t o r s , attempts t o l i n k s t u d i e s on n o n - b i o l o g i c a l adhesion t o n a t u r a l m i c r o b i a l ecology. Growing evidence suggests a c o r r e l a t i o n between b a c t e r i a and microzones of increased n u t r i e n t c o n c e n t r a t i o n . Most organisms adhere to surfaces by means of e x o c e l l u l a r polymers. The composition and qu a n t i t y of polymer has been l i n k e d to environmental c o n d i t i o n s . Rosenberg et a l . (1983a) 24 demonstrated t h a t c e l l s t a r v a t i o n s t r o n g l y decreased the degree of encapsulation of Acinetobacter calcoacetius which i n t u r n increased i t s c e l l surface hydrophobicity. This change i n hydrophobicity markedly e f f e c t e d i t s adhesive p r o p e r t i e s as i l l u s t r a t e d by a reduced adherence to hydrocarbons. I t i s b e l i e v e d that a s u b t l e balance e x i s t s between c e l l surface components which encourage and discourage adherence. The presence of LPS reduced c e l l surface hydrophobicity of Salmonella typhimurium and, as a r e s u l t , decreased i t s adherence to the a i r -water i n t e r f a c e (Hermansson et a l . , 1982). A l t e r n a t i v e l y , the appearance of f i m b r i a e was found to increase c e l l surface hydrophobicity. Adherence s p e c i f i c i t y has been the subject of i n c r e a s i n g debate. For example, Rosenberg et a l . (1983b) demonstrated t h a t the o i l degrading Acinetobacter calcoacetius while adhering t o the o i l -water i n t e r f a c e a l s o r e a d i l y adhered to t o o t h surfaces and e p i t h e l i a l c e l l s . Instead of a s t e r e o - s p e c i f i c component resp o n s i b l e f o r adherence, they argue t h a t these components c o n t r i b u t e to an o v e r a l l hydrophobic c e l l surface which was i n t u r n r e s p o n s i b l e f o r i t s adherence d i v e r s i t y . In 1973, M a r s h a l l and Cruickshank f i r s t drew a c o r r e l a t i o n between c e l l h ydrophobicity and surface adherence. In support of t h i s , Dahlback et a l . (1981) performed hydrophobicity assays on a broad range of b a c t e r i a l i s o l a t e s from both the a i r - w a t e r i n t e r f a c e and subsurface water. Their r e s u l t s suggested that i s o l a t e s w i t h the 25 highest degree of hydrophobicity were a s s o c i a t e d w i t h the a i r -water i n t e r f a c e . Using thermodynamic models, m i c r o b i a l adherence to hydrophobic surfaces i s p r e d i c t a b l y more favourable than adherence to h y d r o p h i l i c surfaces, provided the surface t e n s i o n of the l i q u i d medium i s higher than that of the b a c t e r i a l surface. This i s the case w i t h most n a t u r a l environments and i s supported by experimental data of other workers ( K j e l l e b e r g , 1984) . Various c e l l surface components have been found to act as adhesins. The abundance and composition of these s t r u c t u r e s vary markedly between species and w i t h i n species of microorganisms. The growth c o n d i t i o n s and the p h y s i o l o g i c a l s t a t e of the c e l l can have a s i g n i f i c a n t e f f e c t on the adhesion process. Leech and Heffor (1980) demonstrated an inverse r e l a t i o n s h i p between growth r a t e and S.sanguis d e p o s i t i o n on i n e r t polystyrene l a t e x p a r t i c l e s . Rosan et a l . (1982) f u r t h e r demonstrated t h a t carbohydrate source and growth pH a l s o a f f e c t s S.sanguis adherence. E x t r a c e l l u l a r polymers such as l i p o p o l y s a c c h a r i d e (LPS), of gram negative organisms, and l i p o t e i c h o i c a c i d (LTA) of gram p o s i t i v e organisms can p l a y a c r u c i a l r o l e i n c e l l adherence (Dazzo & Truchet, 1983; Beachey, 1981). LPS mediated adherence i s best demonstrated by the Rhizobium-26 legume symbiosis (Dazzo et a l . , 1982). S p e c i f i c r e v e r s i b l e attachment i s a necessary step towards host c e l l i n f e c t i o n and n i t r o g e n - f i x i n g root module formation. The surface components mediating attachment i n c l u d e a m u l t i v a l e n t l e c t i n ( t r i f o l i i n A) on the root h a i r , and carbohydrate receptors i n the f i b r i l l a r capsule of the Rhizobium c e l l s urface. T r i f o l i i n A l e v e l s on root h a i r s was found t o be regu l a t e d by exogenous n i t r a t e supply (Dazzo & B r i l l , 1978). The l e v e l s of the l e c t i n - b i n d i n g saccharide (quinovosamine) i n Rhizobium LPS i s growth dependent and increases s i g n i f i c a n t l y as c e l l s enter s t a t i o n a r y phase (Hrabak et a l . , 1981). LPS i s h i g h l y d i v e r s e among gram negative organisms. The LPS molecule c o n s i s t s of three d i s t i n c t regions; O-polysaccharides, core polysaccharide and the l i p i d A regi o n . The l i p i d A region i s h i g h l y conserved p o r t i o n of LPS c o n s i s t i n g of phosphorylated glucosamines residues i n the form of o l i g o s a c c h a r i d e s which are attached t o f a t t y a c y l e s t e r s , thus p r o v i d i n g the hydrophobic, membrane bi n d i n g , p o r t i o n of t h i s a m p h i p h i l i c molecule. The core polysaccharide i s al s o h i g h l y conserved and i s c h a r a c t e r i z e d by a l d o h e p t o s e and 2 - k e t o - 3 - d e o x y o c t o n a t e r e s i d u e s . Ketodeoxyoctonate along w i t h s e v e r a l phosphate residues c o n t r i b u t e t o a net negative charge i n t h i s region of the molecule. The O-polysaccharides are polymers of repeating sequences of two t o four mono-saccharides which in c l u d e a wide range of heptoses and pentoses. The h y d r o p h i l i c polysaccharide region of the molecule determines the s e r o t y p i c d i f f e r e n c e s between s t r a i n s of a b a c t e r i a l species (Wicken, 1985). Less s p e c i f i c adherence by surface polymers i s mediated by c e l l surface S l a y e r s or by b a c t e r i a l g l y c o c a l y x . S l a y e r s contain a reg u l a r array of g l y c o p r o t e i n subunits which are a s s o c i a t e d w i t h the c e l l w a l l v i a d i v a l e n t c a t i o n s . The g l y c o c a l y x , or c e l l capsule, i s a h i g h l y hydrated polysaccharide matrix which v a r i e s i n complexity from simple homopolymers to complex heteropolymers. This f i b r o u s matrix may be f l e x i b l e or r i g i d and may be shed by the c e l l or i n t e g r a t e d i n t o the outer membrane. The g l y c o c a l y x i s found i n a v a r i e t y of n a t u r a l ecosystems and i s ofte n l o s t upon i n v i t r o s u b c u l t u r i n g . During the attachment process the gl y c o c a l y x i s thought to overcome e l e c t r o s t a t i c r e p u l s i v e f o r c e s , experienced by the b a c t e r i a , by forming a bridge between the c e l l and the surface. Once contact i s e s t a b l i s h e d , weaker forces c o n t r i b u t e towards an i r r e v e r s i b l e adherent s t a t e (Costerton, 1981) . This view has been disputed by Sutherland (1983), suggesting t h a t the g l y c o c a l y x may not be i n v o l v e d i n i n i t i a l attachment, but plays a greater r o l e i n e s t a b l i s h i n g i r r e v e r s i b l e attachment. Prosthecate b a c t e r i a , such as Caulobacter spp., are c h a r a c t e r i z e d by p o l a r extensions, termed s t a l k s . The d i s t a l p o r t i o n of the b a c t e r i a l s t a l k contains a h o l d f a s t adhesive which i s re s p o n s i b l e f o r adherence t o surfaces. The l i f e c y c l e of Caulobacter i s 28 separated by a swarmer c e l l phase and a surface attached phase. The h o l d f a s t , which i s present during both phases of the dimorphic l i f e c y c l e , provides a good system f o r studying the molecular mechanisms of adhesion. C h a r a c t e r i z a t i o n of Caulobacter adherence i s c u r r e n t l y being i n v e s t i g a t e d (Merker & Smit, 1988) . Fimbriae and p i l i are the most widely s t u d i e d c e l l surface appendages. Fimbriae i s o l a t e d from gram negative organisms are short, s t r a i g h t p r o t e i n appendages, made up of subunits which vary from 15 t o 25 Kd (Jones & Isaacson, 1983) . The amino terminus i s h i g h l y conserved and s t r o n g l y hydrophobic suggesting a p o s s i b l e a s s o c i a t i o n w i t h the outer membrane. They are most prominent i n gram negative b a c t e r i a and have been i m p l i c a t e d i n adhesion and c e l l aggregation. The adherence process has a l s o i n v o l v e d the p i l u s . P i l i , u n l i k e f i m b r i a e , are plasmid-encoded lar g e proteinaceous appendages which f a c i l i t a t e t r a n s f e r of genetic m a t e r i a l between donor and r e c i p i e n t c e l l s . Another c e l l surface s t r u c t u r e i n v o l v e d i n adherence are f i b r i l s . Handley et a l . (1985) has described f i b r i l s on a number of gram p o s i t i v e and gram negative organisms. U n l i k e f i m b r i a e , these r a d i a t i n g s t r u c t u r e s do not have a defined width. Duguid et a l . (1955) f i r s t i d e n t i f i e d the n o n - f l a g e l l a r , p o l a r and p e r i t r i c i o u s , short filamentous appendages i n Escherichia coli. These surface s t r u c t u r e s , designated antigen K-88 and K-99 have been shown to mediate E.coli d i a r r h o e a l disease i n p i g l e t s and c a l v e s , r e s p e c t i v e l y (Orskov et a l . , 1961; Orskov et a l . , 1975) . I t i s b e l i e v e d these antigens play a r o l e i n attachment le a d i n g t o v i r u l e n c e since i n f e c t i v i t y i s i n h i b i t e d by adhesin s p e c i f i c a n t i s e r a or p u r i f i e d f i m b r i a e . In a d d i t i o n , non-f i m b r i a t e d s t r a i n s of E.coli were found to be n o n - v i r u l e n t . Since K88 and K99 were shown to be plasmid encoded, they have been c a t e g o r i z e d as p i l i i n s t e a d of fimbriae (Isaacson, 1984). This phenomenon has been observed i n other gram negative organisms. P i l i - m e d i a t e d attachment of pathogenic Neisseria spp. t o mucosal surfaces has been demonstrated (Stephens, 1984). Of a l l the adherence mechanisms discussed, t h i s i s an example of the most s p e c i a l i z e d form since a s p e c i f i c i n t e r a c t i o n between the p i l i and the receptor molecule on the i n t e s t i n a l e p i t h e l i u m i s necessary f o r attachment. A number of d i f f e r e n t techniques have been employed i n the study of m i c r o b i a l adherence. These methods can be d i v i d e d i n t o two general approaches: A) C h a r a c t e r i z a t i o n of the adherence r e a c t i o n . B) 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 the adhesin. Adherence r e a c t i o n c h a r a c t e r i z a t i o n can i n v o l v e chemical or enzymatic m o d i f i c a t i o n of the c e l l surface. For example, protein-mediated adherence can be e l i m i n a t e d by t r e a t i n g the c e l l s w i t h a protease (Weerkamp et a l . , 1980). S i m i l a r l y , s i a l i c a c i d residue removal by neuraminidase was shown to e f f e c t 30 Streptococcus sanguis i n t e r a c t i o n w i t h s a l i v a r y p r o t e i n s (McBride et a l . , 1977; Levine et a l . , 1978). Chemical treatment of c e l l s can a l s o markedly e f f e c t adherence. For example, treatment w i t h reducing agents i m p l i e d the involvement of • s u l f h y d r y l groups i n the adherence of Sulfolobus to sulphur (Wiess, 1973). Competitive i n h i b i t i o n s t u d i e s using receptor analogues to c h a r a c t e r i z e adherence i s a common approach. Carbohydrate-s e n s i t i v e adherence has been described i n o r a l b a c t e r i a by Duguid et a l . (1966). Competitive b i n d i n g experiments between s i m i l a r or d i f f e r e n t c e l l types has been a u s e f u l t o o l i n studying adherence. Staat et a l . (1984), used competitive b i n d i n g between r a d i o a c t i v e and non-radioactive c e l l s t o d i s t i n g u i s h s p e c i f i c from n o n - s p e c i f i c adherence. Adsorption isotherms have become a u s e f u l t o o l f o r studying adherence s p e c i f i c i t y and k i n e t i c s . The b i n d i n g isotherm i s obtained by p l o t t i n g the number of f r e e c e l l s (U) versus the number of bound c e l l s (B) at e q u i l i b r i u m i n a given system. Information about the adherence k i n e t i c s can be obtained using the Langmuir equation; U/B = K/N + (1/N)U, where K i s the d i s s o c i a t i o n constant and N i s the maximum number of b i n d i n g s i t e s . By p l o t t i n g U/B verses U, an e s t i m a t i o n of the d i s s o c i a t i o n constant and the a f f i n i t y constant, (1/K from the x i n t e r c e p t ) , can be determined. Though the c o e f f i c i e n t of v a r i a t i o n i s high using these isotherms, some u s e f u l i n f o r m a t i o n regarding c e l l - s u r f a c e i n t e r a c t i o n s can be obtained (Gibbons et a l . , 1976) . The more d i f f i c u l t method of studying adherence i s through the 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 the c e l l adhesin. A popular approach used by many workers i s the i s o l a t i o n of non-adherent mutants. The c e l l surface p r o t e i n r e s p o n s i b l e f o r adherence can i n some cases, be i d e n t i f i e d by comparing the outer membrane p r o f i l e s of the mutant and the wild-type (Fives-Taylor et a l . , 1985; Gaastra et a l . , 1982). Mutants l a c k i n g a surface adhesin can then be used t o adsorb wild-type a n t i s e r a i n order to p u r i f y monospecific p o l y c l o n a l a n t i s e r a t o the adhesin molecule (Fachon-Kalweit et a l . , 1985; C i s a r et a l . , 1983). The a d h e s i n - s p e c i f i c a n t i s e r a i s u s e f u l i n adhesin l o c a l i z a t i o n s t u d i e s through immunogold techniques and i n adherence i n h i b i t i o n s t u d i e s . Several researchers have addressed the question whether attached b a c t e r i a c o n t r i b u t e t o the s u r v i v a l of f r e e - l i v i n g b a c t e r i a . I t i s g e n e r a l l y accepted t h a t i n a n u t r i e n t depleted aquatic environment, organic substrates tend t o accumulate at l i q u i d i n t e r f a c e s . However, i n n a t u r a l aquatic environments, only a small percent of the t o t a l surface area i s c o l o n i z e d by b a c t e r i a (Hoppe, 1984). K h a i l o v and co-workers (1970) made three observations which could e x p l a i n t h i s environmental enigma: 1) High molecular weight macromolecules which are prevalent 32 i n n a t u r a l aquatic systems and d i s p l a y high degrees of surface a c t i v i t y , are almost e x c l u s i v e l y adsorbed t o surfaces. 2) Attached b a c t e r i a e x h i b i t higher e x t r a c e l l u l a r enzymatic c a p a c i t i e s necessary f o r macromolecule degradation than t h e i r f r e e - l i v i n g counterparts. 3) Attached b a c t e r i a do not immediately consume t h e i r products r e s u l t i n g from macromolecular degradation. They i n t e r p r e t these f i n d i n g s t o mean that attached b a c t e r i a b e n e f i t the f r e e - l i v i n g b a c t e r i a by degrading the high molecular weight macromolecules and make a v a i l a b l e s o l u b l e organic s u b s t r a t e s . I w i l l now review the current methods of measuring m i c r o b i a l adherence t o mineral surfaces (Van Es & Meyer-Reil, 1982). Most i n v e s t i g a t o r s have measured c e l l adherence i n d i r e c t l y and determined the concent r a t i o n of unbound c e l l s by c o r r e l a t i n g c e l l numbers wi t h v a r i o u s biomass or metabolic i n d i c a t o r s . Examples of biomass measurement i n d i c a t o r s are: 1) P r o t e i n concentration (Myerson et a l . , 1983; D i S p i r i t o et a l . , 1983). 2) O p t i c a l d e n s i t y measurements at 660 nm (Kakakuwa et a l . , 1979) . 3 3 3) Organic n i t r o g e n concentration (Gormely et a l . , 1974). 4) Adenosine triphosphate (ATP) content (Tuovinen & Sormuneu, 1979). B i o a c t i v i t y i n d i c a t o r s used to measure adherence are: 1) Radioactive carbon f i x a t i o n ( B r i e r l e y , 1977). 2) Radioactive phosphate i n c o r p o r a t i o n (McCready & L e G a l l a i s 1984). The major drawback to i n d i r e c t techniques i s t h a t one cannot d i s t i n g u i s h the b i n d i n g of one c e l l type from another. In a d d i t i o n , microscopic mineral f i n e s , which i n t e r f e r e w i t h many biomass assays, are d i f f i c u l t t o remove from unbound c e l l s l e f t i n suspension. P r o t e i n and n i t r o g e n c o n c e n t r a t i o n assays r e s t r i c t the p o s s i b l e uses of c e r t a i n p r o t e i n or n i t r o g e n -c o n t a i n i n g adherence i n h i b i t o r s . R adioactive isotope s t u d i e s have problems of decay quenching during s c i n t i l l a t i o n counting. ATP assay techniques have encountered d i f f i c u l t i e s w i t h ATP e x t r a c t i o n and measurement i n the presence of high l e v e l s of i r o n compounds and heavy metals i n the samples. The i n t r o d u c t i o n of f l u o r e s c e n t - a n t i b o d y (FA) techniques allowed d i r e c t observation of bound c e l l s (Apel et a l . , 1976; Muyzer et a l . , 1987). In a d d i t i o n , f l u o r e s c e n t - a n t i b o d y microscopy can i d e n t i f y s p e c i f i c c e l l s t r a i n s , p r o v i d i n g the antibody used i s s p e c i f i c . The FAINT technique (Baker & M i l l s , 1982), which i s a modified v e r s i o n of the FA technique, allows the measurement of a c t i v e l y growing c e l l s through the b i o l o g i c a l r e d u c t i o n of I N T ( 2 - ( p - i o d o p h e n y l ) - 3 - ( p - n i t r o p h e n y l ) - 5 phenyl t e t r a z o l i u m c h l o r i d e ) to a pigmented formazan compound which accumulates i n the c e l l . This type of p h y s i o l o g i c a l i n f o r m a t i o n can a l s o be obtained through e p i f l u o r e s c e n c e microscopy (EFM) using a c r i d i n e orange (Yeh et a l . , 1987) which, depending on the a c t i v i t y of the c e l l , w i l l f l u o r e s c e d i f f e r e n t c o l o u r s . However, c o n f l i c t i n g r e s u l t s have been experienced by the l a t t e r two techniques; both authors a t t r i b u t e d these i n c o n s i s t e n c i e s to the unknown e f f e c t s of low pH. Though these d i r e c t microscopic techniques can be i n f o r m a t i v e , FAINT and EFM are labour i n t e n s i v e . In a d d i t i o n , background fluorescence through n o n - s p e c i f i c adsorption by the antibody t o the mineral surface, can i n t e r f e r e w i t h adherence e s t i m a t i o n s . In l i g h t of the drawback discussed regarding current adherence assays, I have developed a n o n - i s o t o p i c , s p e c i f i c , s e n s i t i v e approach to measuring m i c r o b i a l adherence to mineral surfaces. Growing evidence supports the importance of substrate attachment i n i n d u s t r i a l a p p l i c a t i o n s of a c i d o p h i l i c microorganisms; however, a thorough a n a l y s i s of a c i d o p h i l e attachment mechanisms i s l a c k i n g . Given the importance of h e t e r o t r o p h i c a c i d o p h i l e s and t h e i r symbiotic r e l a t i o n s h i p w i t h a u t o t r o p h i c a c i d o p h i l e s , I have l i m i t e d my study p r i m a r i l y to the organism, Acidiphilium 35 cryptum. The purpose of t h i s report then i s to provide a comprehensive study of h e t e r o t r o p h i c a c i d o p h i l e adherence t o mineral surfaces by c h a r a c t e r i z i n g the adherence r e a c t i o n and studying adherence-defective mutants. 36 MATERIALS AND METHODS Bacteria The b a c t e r i a l s t r a i n s used i n t h i s study were A.cryptum, ATCC 33463, and T.ferrooxidans, ATCC 23270. Both c u l t u r e s were obtained from American Type Culture C o l l e c t i o n . Media and stock maintenance A.cryptum was c u l t i v a t e d on a medium c o n t a i n i n g the f o l l o w i n g per l i t r e : (NH 4) 2S0 4, 2 gm; KC1, 0.1 gm; K2HP04, 0.25 gm; MgS04-7H20, 0.25 gm; Ca (N03) 2-4H20, 0.01 gm; dehydrated yeast e x t r a c t (Difco l a b o r a t o r i e s , Detroit,Michigan) , 0.1 gm; D-dextrose, 1.0 gm. This complete media w i l l be r e f e r r e d to as MSGYE medium f o r the re s t of the t e x t . Media without the organic i n g r e d i e n t s was designated b a s a l mineral s a l t s . The ba s a l mineral s a l t s was pH adjusted t o 3.2 wi t h 1 N H2S04. To prepare MSGYE media, double strength b a s a l mineral s a l t s (pH 3.2) and double stre n g t h glucose-yeast e x t r a c t s o l u t i o n s were autoclaved separately at 15 l b / i n 2 f o r 20 min and then a s e p t i c a l l y mixed a f t e r c o o l i n g . For s o l i d media, 15 g m / l i t r e of agar was added t o the glucose-yeast e x t r a c t s o l u t i o n p r i o r to au t o c l a v i n g . Small, white, convex, round c o l o n i e s could be detected w i t h i n three days of growth at 30° C. A.cryptum was stor e d on MSGYE agar s l a n t s at 4° C f o r one month periods between t r a n s f e r s . Stock c u l t u r e s of A.cryptum were maintained at -70° C i n a ba s a l mineral s a l t s s o l u t i o n c o n t a i n i n g 10% g l y c e r o l (pH 3.2). Late log-phase growing c e l l s were harvested from a 3 day c u l t u r e incubated at 30° C. P l a t e -grown c e l l s were ge n t l y removed from the agar surface and washed once i n 1/2 concentration b a s a l mineral s a l t s (1/2 MS), pH 3.2 which w i l l be r e f e r r e d to as adherence b u f f e r . T.ferrooxidans was grown and maintained on 100:10 s o l i d media as described by Schrader and Holmes (1988) . E x p o n e n t i a l l y growing c e l l s were harvested from a 7 day c u l t u r e grown at 30° C and washed once i n adherence b u f f e r . C e l l suspension concentrations were determined using a Petroff-Hausser counting chamber and phase-contrast microscopy. Buffers The adherence b u f f e r c o n s i s t e d of a 1/2 d i l u t i o n of the b a s a l mineral s a l t s which was adjusted to pH 3.2 w i t h 1 N H2S04. For i n h i b i t i o n assays, the v a r i o u s p u t a t i v e i n h i b i t o r s were added to the adherence b u f f e r . Ore ELISA b u f f e r s , PBS/Tween and PBS 1% BSA, were each pH adjusted to 5.0 w i t h 1 N HCl. Bio-Rad Adherence Assay A known number of c e l l s were resuspended i n adherence b u f f e r . 1.5 ml of the c e l l suspension was added to microfuge tubes c o n t a i n i n g pre-measured amounts of mineral ore. This mixture was a g i t a t e d through continuous i n v e r s i o n by the Labquake (L a b i n d u s t r i e s , C a l i f . ) at 20° C f o r the d u r a t i o n of the i n c u b a t i o n p e r i o d . The ore was removed from s o l u t i o n by low 38 speed c e n t r i f u g a t i o n (5800xg) f o r 20 seconds. To c o n t r o l f o r f a l s e p o s i t i v e c e l l l o s s during t h i s step, "spun" and "non-spun" c e l l suspension c o n t r o l s were performed. A 0.9 ml volume of the supernatant, c o n t a i n i n g the unbound c e l l s l e f t i n suspension, was removed to a new microfuge tube. The unbound c e l l s were harvested by high speed c e n t r i f u g a t i o n (13, OOOxg) f o r 10 min. The supernatant was discarded and the c e l l p e l l e t was resuspended i n d i s t i l l e d water. The c e l l s were then t r a n s f e r r e d t o gl a s s tubes and b o i l e d f o r 10 min to l y s e the c e l l s . When the tubes were c o o l , 0.2 ml of Bio-Rad p r o t e i n assay concentrate (Bio-Rad L a b o r a t o r i e s , Richmond, CA.) was added to each tube and p r o t e i n c oncentration was measured at A 5 g s. In order t o c a l c u l a t e adherence, the f o l l o w i n g equation was used: % Adherence = [ 1 - ( t e s t / c e l l spun c o n t r o l )] x 100 The number of c e l l s bound was determined by m u l t i p l y i n g the % adherence by the number of c e l l s o r i g i n a l l y added to the assay tube. Ore ELISA Adherence Assay This assay procedure i s a modified v e r s i o n of the Enzyme-Linked Immunoadsorbent Assay (ELISA), by E n g v a l l (1972). To prevent c e l l adsorption t o p r e p a r a t i v e m a t e r i a l s , a l l glassware was s i l i c o n i z e d w i t h Surfa S i l (Pierce Chem. Co., Rockford, I I . ) . M i n e r a l ore samples were provided by B r i t i s h Columbia Research 39 I n s t , and are d e s c r i b e d i n Table I. The ore samples were washed, des i c c a t e d and f i n e l y ground to an average p a r t i c l e s i z e of 1.5 yum. A coat of s i l i c o n e - b a s e d glue (Dow Corning corp., Midland, MI.) was a p p l i e d t o each w e l l of a fl a t - b o t t o m m i c r o t i t e r p l a t e (Gibco, Canada I n c . ) . Each w e l l was then coated w i t h prepared mineral ore and allowed t o dry overnight (Fig. 5) . Unbound ore was removed and the p l a t e was thoroughly r i n s e d w i t h d i s t i l l e d water t o remove l o o s e l y bound p a r t i c l e s . The p l a t e s were then d r i e d at 37° C f o r one hour. A known number of c e l l s were resuspended i n adherence b u f f e r . 300 yul of c e l l suspension was added to each w e l l and incubated on a r o t a r y shaker (100 rpm) at 20° C. A f t e r the d e s i r e d p e r i o d of Table I. T i t l e and d e s c r i p t i o n of mineral ore. Source Geological constituent Newmont mine chalcopyrite/pyrite Gibraltar mine chalcopyrite/pyrite Empire mine pyrite Cambell Red lake pyrite/arsenopyrite 40 i n c u b a t i o n , the unbound c e l l s were removed and each w e l l was r i n s e d twice w i t h adherence b u f f e r . 400 /AI of b l o c k i n g s o l u t i o n (PBS/1% BSA, pH 5.0) was added to each w e l l and incubated f o r one hour at 37° C. A f t e r the b l o c k i n g s o l u t i o n was removed, the p l a t e was r i n s e d twice w i t h washing b u f f e r (PBS/Tween, pH 5.0). The primary antibody was d i l u t e d i n PBS/1% BSA (pH 5.0) and 300 >*1 was added to each w e l l and incubated overnight at 4° C. The primary antibody was removed and the p l a t e was washed 3 times wi t h washing b u f f e r (the l a s t wash was allowed to s i t f o r 10 min). The secondary antibody s o l u t i o n contained a l k a l i n e phosphatase conjugated goat a n t i - r a b b i t IgG (Helix B i o t e c h l t d . ) d i l u t e d i n PBS/1% BSA (pH 5.0). 300 >»1 of secondary antibody s o l u t i o n was added to each w e l l and incubated at 37° C f o r one hour. The secondary antibody was removed and the p l a t e s were washed 3 times w i t h washing b u f f e r (the l a s t wash was again allowed t o s i t f o r 10 min). The p l a t e s were developed by adding 300 /AI of a l k a l i n e phosphatase substrate (Sigma Chemical Co., St.Louis, Mo.) to each w e l l and in c u b a t i n g at 37° C i n the dark. Upon colour development, 100 ,M1 of spent a l k a l i n e phosphatase substrate was removed t o a second m i c r o t i t e r p l a t e f o r adsorbance reading at 405 nm. In order t o c a l i b r a t e the A 4 0 5 values to b a c t e r i a per m i l l i l i t r e , s p e c i f i c a c t i v i t y measurements were performed f o r each assay run. The antibody r e a c t i v i t y was determined by measuring the A 4 0 5 values of known c e l l c o ncentrations, i n 1 ml volumes, under i d e n t i c a l c o n d i t i o n s as the Ore ELISA assay. This assay was performed i n microfuge tubes t o allow the c e n t r i f u g a t i o n of the c e l l s between each assay step. Isolation of adherence variants Ore adherence v a r i a n t s were obtained through n i t r o s o g u a n i d i n e (NG) mutagenesis (Adelberg, 1965) followed by s e v e r a l enrichment steps. The Ore ELISA (as described above) was used to screen the enriched c e l l p o p u l a t i o n f o r adherence-defective mutants. A s u r v i v a l curve was determined w i t h a constant c o n c e n t r a t i o n of NG and d i f f e r e n t time exposures. E x p o n e n t i a l l y growing c e l l s were harvested washed and resuspended i n c i t r a t e b u f f e r to an OD660 of 0.5. NG was added to a f i n a l c o n c e n t r a t i o n of 50 /^g/ml and t h i s was incubated at 37° C. At 20 min i n t e r v a l s c e l l s were removed, washed and resuspended i n phosphate b u f f e r and p l a t e d out on MSGYE media to determine the number of colony forming u n i t s (CFU). Mutagenized c e l l s were incubated f o r 24 hrs i n MSGYE l i q u i d media. The c e l l s were washed and resuspended i n adherence b u f f e r p r i o r t o enrichment. Each enrichment procedure c o n s i s t e d of two adsorption steps. The adsorption step i n v o l v e d a s e p t i c a l l y mixing 20 mis of the c e l l suspension (approx. l x l O 9 c e l l s per ml) with 1 gm of s t e r i l e Newmont mineral ore. The mixture was a g i t a t e d through continuous i n v e r s i o n i n a labquake f o r 20 min at 20° C. A f t e r i n c u b a t i o n the ore was removed by d i f f e r e n t i a l c e n t r i f u g a t i o n (2 min at 500xg) and the supernatant was recovered. A f t e r repeating once, the unbound c e l l s were resuspended i n MSGYE media and grown f o r three days at which time the next enrichment was performed. When enrichment was completed the c e l l s were p l a t e d and examined m a c r o s c o p i c a l l y and m i c r o s c o p i c a l l y . Selected c e l l s were screened f o r adherence a b i l i t y using the Ore ELISA assay, the Bio-Rad assay and a second i n d i r e c t method described by Takakuwa et a l . (197 9) i n which biomass i s measured by OD660 . C e l l envelope protein preparation C e l l s were washed i n adherence b u f f e r and resuspended i n a b u f f e r c o n t a i n i n g 0.05 M sodium phosphate, 0.15 M sodium c h l o r i d e and 0.01 M EDTA adjusted to pH 7.4 (Boyd & McBride, 1984). The c e l l suspension was sonicated (Branson Sonic Power Co., Danbury, Conn.) 3 times f o r 40 sec on pulsed mode followed by 10 sec on continuous mode. . The mixture was then c e n t r i f u g e d at 10,000*g f o r 20 min to remove unbroken c e l l s . The supernatant was recovered and c e n t r i f u g e d at 80,000xg f o r 2 hours. The p e l l e t of c e l l envelope p r o t e i n (CEP) was resuspended i n d i s t i l l e d water and i t s concentration was determined by the Bio-Rad p r o t e i n assay. C e l l envelope s o l u b i l i z a t i o n P r o t e i n s were s o l u b i l i z e d from the CEP pr e p a r a t i o n using v a r i o u s detergents; 0.1% Chaps, 0.1% T r i t o n X-100, 1 M l i t h i u m c h l o r i d e , 8 M urea, 5 M sodium thiocyanate, 6 M guanidine HCl, 0.1 M EDTA, 2.0% SDS or a combination of 8 M urea, 6 M guanidine HCl and 0.1 M EDTA. 50 j^q of CEP was added to 2 ml of each of the detergents. This mixture was incubated f o r 2 hours at 37° C and c e n t r i f u g e d f o r 2 hours at 80,000xg. The supernatant was d i a l y s e d overnight at 4° C against d i s t i l l e d water. A sample of CEP p r e p a r a t i o n was t r e a t e d w i t h d i s t i l l e d water and a f t e r c e n t r i f u g a t i o n , the supernatant and p e l l e t were separated to act as negative and p o s i t i v e c o n t r o l s r e s p e c t i v e l y . I n h i b i t i o n assay C e l l s were p r e t r e a t e d w i t h v a r i o u s agents and t e s t e d , by the Ore ELISA, t o determine t h e i r e f f e c t on c e l l adherence. These agents in c l u d e d ; p o l y s a c c h a r i d e s , p r o t e i n s , monovalent and d i v a l e n t c a t i o n s , reducing agents, b a c t e r i c i d a l agents, metal c h e l a t i n g agents and surface a c t i v e agents. C e l l s were washed and resuspended, to a concent r a t i o n of l x l O 9 c e l l s per ml, i n adherence b u f f e r c o n t a i n i n g one of the agents. The b a c t e r i a were incubated f o r 30 min at 20° C before dispensing 300 yul t o each Ore ELISA p l a t e w e l l . C e l l modification C e l l s were subjected t o enzymatic, p h y s i c a l and heat treatment to i n v e s t i g a t e t h e i r e f f e c t on adherence to min e r a l . The c e l l s were washed and resuspended, t o a concentration of l x l O 9 c e l l s per ml, 44 i n each treatment b u f f e r . Enzymatic treatment b u f f e r (50 mM T r i s , pH 7.2) contained e i t h e r ; 50 yi/g/ml phospholipase C, 50 yug/ml proteinase K, or 100 /^g/ml mixed g l y c o s i d a s e . Phospholipase C b u f f e r was supplemented w i t h 1 mM CaCl 2. For enzymatic treatment, c e l l s were incubated f o r 1 hr at 37° ,C. Heat treatment i n v o l v e d i n c u b a t i o n f o r ; 60 min at 50° C, 10 min at 70° C or 5 min at 100° C, i n 50 mM T r i s b u f f e r , pH 7.2. P h y s i c a l treatment i n v o l v e d v o r t e x i n g c e l l s w i t h a magnetic s t i r bar f o r 30 min i n 50 mM T r i s , pH 7.2 at 20° C. A f t e r treatment, c e l l s were washed and resuspended i n adherence b u f f e r f o r assay. Competition assay Competitive b i n d i n g assays were preformed w i t h immunologically d i s t i n c t c e l l types. The two c e l l types were mixed p r i o r to adding them to the Ore ELISA p l a t e . One c e l l type was maintained at a constant concentration while the other c e l l type was added i n i n c r e a s i n g c o ncentrations. The primary antibody used during the Ore ELISA assay was d i r e c t e d towards the c e l l s which were kept at constant c o n c e n t r a t i o n . Hydrophobicity assay B a c t e r i a l surface hydrophobicity was determined by measuring the number of b a c t e r i a adhering to hexadecane (Rosenberg et a l . , 1980; Ganeshkumar, 1985) . C e l l s were washed and resuspended i n adherence b u f f e r t o an absorbance at A 6 6 0 of 0.5. A volume of 0.1 ml hexadecane was added to 3.0 ml of c e l l suspension i n a 18x150 mm t e s t tube. The suspension was mixed on a vortex mixer f o r 60 sec at 10 sec i n t e r v a l s and allowed to stand f o r 20 min before reading. The c e l l s remaining i n the aqueous phase were c a r e f u l l y removed and A 6 6 0 values were determined. The t e s t samples were compared to c o n t r o l samples which were not t r e a t e d w i t h hexadecane. Surface protein preparation (SPP) C e l l s were washed and resuspended, t o a con c e n t r a t i o n of 5 x l 0 9 c e l l s per ml, i n a b u f f e r c o n t a i n i n g ; 20 mM T r i s , 150 mM NaCl, 10 mM MgCl 2, pH 7.2. This s o l u t i o n was mixed wi t h a magnetic s t i r bar f o r 30 min at 20° C. The c e l l s were removed by c e n t r i f u g a t i o n and the supernatant recovered. Forty percent ammonium sulphate was added to the supernatant and slowly s t i r r e d overnight at 4° C. The p r e c i p i t a t e was p e l l e t e d (80,000xg f o r 2 hrs) and resuspended i n d i s t i l l e d water. The pr e p a r a t i o n was ex h a u s t i v e l y d i a l y s e d against d i s t i l l e d water at 4° C and frozen at -20° C. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE, as described by Laemmli (1970), was performed w i t h a 10% or 12% polyacrylamide separating g e l and a 3% s t a c k i n g g e l . The samples were b o i l e d f o r 10 min i n SDS-^ME before adding approximately 0.3 y- o E >. v> O < 0 5 10 15 20 25 T.ferrooxIdans/A.cryptum F i g u r e 12. Competition between A.cryptum and T. ferrooxidans f o r b i n d i n g s i t e s on c h a l c o p y r i t e : a) Increasing numbers of A.cryptum c e l l s were added t o a constant number of T.ferrooxidans c e l l s (2xl0 9 c e l l s / m l ) . b) Increasing numbers of T. ferrooxidans c e l l s were added to a constant number of A.cryptum c e l l s (3xl0 9 c e l l s / m l ) . 80 A.cryptum t o c h a l c o p y r i t e i n the presence of an excess of T. ferrooxidans ( F i g . 12 (b)) . I t was concluded t h a t the b i n d i n g s i t e s f o r each organism are unique and d i s t i n c t . Isolation of adherence-defective variants In order to i d e n t i f y the adhesin r e s p o n s i b l e f o r attachment to mineral surfaces, A.cryptum c e l l s were mutagenized, enriched and screened f o r adherence-defective mutants. Each of three mutagenesis experiments performed used a s t a r t i n g c o n c e n t r a t i o n of 3 x l 0 8 c e l l s per ml and 50 / A g / m l of n i t r o s o g u a n i d i n e . The f i r s t mutagenesis was aimed at a 50% k i l l according to the s u r v i v a l curve ( F i g . 13). This r e q u i r e d a 47 min exposure to the mutagen and was followed by a s e r i e s of 6 enrichment procedures. In a second and t h i r d experiment c e l l s were exposed to NG f o r 80 min and enriched f o r non-adherent c e l l s . C e l l s were enriched 6 and 14 times f o l l o w i n g second and t h i r d enrichments r e s p e c t i v e l y . In these experiments, enrichment i n v o l v e d mixing mutagenized c e l l s w i t h ore f o r 20 min and then c u l t u r i n g c e l l s which d i d not s e t t l e w i t h the ore. The f i r s t two attempts at o b t a i n i n g non-adherent mutants were unsuccessful. Assuming t h a t b i n d i n g may i n v o l v e more than one type of receptor-adhesin i n t e r a c t i o n , i t was decided to modify the t h i r d mutagenesis enrichment procedure i n an attempt t o d i f f e r e n t i a t e between weak and strong b i n d i n g 81 0 H r—i J l i 1 1 r—] r — i 1 1 • 1 • • 1 0 20 40 60 80 100 120 Time (min) Figure 13. N i t r o s o g u a n i d i n e s u r v i v a l curve. 82 c e l l s . Instead of a 20 min i n c u b a t i o n w i t h 1 gm of Newmont ore, the c e l l suspension was incubated w i t h e i t h e r 25 mg of ore f o r 20 min, or 100 mg of ore f o r 10 sec. The modified enrichments would s e l e c t f o r b i n d i n g strength and b i n d i n g speed r e s p e c t i v e l y . These enrichments were performed 6 times, the c e l l s remaining i n suspension were p l a t e d and c o l o n i e s screened f o r t h e i r a b i l i t y to b i n d t o c h a l c o p y r i t e . I s o l a t e s were screened by the Ore ELISA and recorded as the percent of adherent c e l l s r e l a t i v e t o the number of parent c e l l s bound. I n d i r e c t assays confirmed the Ore ELISA screening r e s u l t s . P r i o r t o screening, the c e l l c o n c e n t r a t i o n of the mutant and parent s t r a i n s was adjusted to l x l O 9 c e l l s per ml. Each mutant was t e s t e d f o r antibody r e a c t i v i t y by the tube assay to ensure that mutagenesis d i d not create q u a n t i t a t i v e changes i n the r e a c t i v i t y of the c e l l s to the antibody. Several of the mutants proved to be s e l f - a g g r e g a t i n g and were not f u r t h e r s t u d i e d . Table X l i s t s s e v e r a l of the mutants i s o l a t e d . A s t r i c t l y non-adherent mutant was not obtained. Most mutants d i s p l a y e d a 41-61% decrease i n a b i l i t y t o adhere to c h a l c o p y r i t e . However, mutant-4 showed an increase i n the a b i l i t y to b i n d t o c h a l c o p y r i t e . Mutants -30, -39, -76, and -90 were i s o l a t e d from 83 Table X. C h a r a c t e r i s t i c s of adherence-defective mutants. Strain Adhe rence 1 Hydrophobic i ty 2 Self-aggregation Parental 100 86 -4 128 85 _ 11 40 83 -12 51 78 -15 51 89 -19 43 65 + 23 40 69 + 30 52 77 -39 58 48 + 76 59 58 + 90 39 40 + 1. Percen t of ce l ls bound relative to the control . 2. Percent of cel ls remaining in the aqueous phase. 84 the f i r s t mutagenesis, the t h i r d mutagenesis, any of the i s o l a t e s . The remaining mutants were i s o l a t e d from Phenotype r e v e r s i o n was not detected i n Analysis of adherence-defective mutants Many studie s have c o r r e l a t e d c e l l surface hydrophobicity w i t h adherence t o mineral surfaces and to i n t e r f a c e s i n the environment. However, i n these s t u d i e s , a r e l a t i o n s h i p between c e l l surface hydrophobicity and the a b i l i t y to b i n d to ore was not observed. For example, mutant -90 i s hydrophobic and mutant-15 i s h y d r o p h i l i c yet both are d e f e c t i v e i n t h e i r a b i l i t y to adhere t o c h a l c o p y r i t e . Hydrophobic c e l l s appeared t o s e l f -aggregate . Many microorganisms a t t a c h to surfaces v i a p o l a r or l a t e r a l f l a g e l l a . This d i d not appear to be the case w i t h A. cryptum since the adherence-defective mutants possessed f l a g e l l a and are m o t i l e . Western immunoblot a n a l y s i s of whole c e l l l y s a t e s showed th a t a 31.6 kDa antigen present i n the parent s t r a i n ( F i g . 14, Lane A), was absent from the adherence-defective mutants ( F i g . 14, Lanes D, E, & F ) . Mutant-4 ( F i g . 14, Lane C) which showed enhanced 8 5 Figure 14. Western b l o t o f a 10% SDS-PAGE d e v e l o p e d w i t h anti - A . c r y p t u m a n t i b o d y . Whole c e l l l y s a t e s o f : Lane A and B, p a r e n t a l A.cryptum. Lane C, mutant-4 which showed enhanced adherence a b i l i t y . Lanes D, E, and F are a d h e r e n c e - d e f e c t i v e mutants-11,-12, and -15 r e s p e c t i v e l y . Samples i n l a n e s A, C-F, were h e a t e d t o 100° C i n SDS p r i o r t o e l e c t r o p h o r e s i s . The sample i n l a n e B was i n c u b a t e d i n SDS a t 20° C p r i o r t o e l e c t r o p h o r e s i s . 86 adherence a b i l i t y , a l s o contained the 31.6 kDa antigen. B o i l i n g the sample had no e f f e c t on the migra t i o n p a t t e r n of the 31.6 kDa antigen ( F i g . 14, Lane A & B) . An a d d i t i o n a l low molecular weight antigen, common to a l l mutants, was i d e n t i f i e d i n Figure 14. The antigen m i g r a t i o n p r o f i l e of each mutant c l o s e l y resembles the p a r e n t a l s t r a i n , suggesting that the mutants are not contaminants. This was supported by microscopic and macroscopic comparisons. The 31.6 kDa p r o t e i n was absent i n a l l the adherence-defective mutants t e s t e d . The p r o t e i n was present i n p a r e n t a l c e l l s harvested from l i q u i d and s o l i d media. C e l l s from e i t h e r medium had equivalent ore adherence a b i l i t i e s . L o c a l i z a t i o n of the 31.6 kDa protein Since the 31.6 kDa p r o t e i n was absent i n adherence-defective s t r a i n s , and present i n p a r e n t a l and adherence-enhanced s t r a i n s , i t was p o s t u l a t e d that the p r o t e i n might be i n v o l v e d i n the bin d i n g of A.cryptum to c h a l c o p y r i t e . Therefore, attempts were made t o l o c a l i z e the 31.6 kDa p r o t e i n . On the assumption that l o o s e l y a s s o c i a t e d c e l l surface s t r u c t u r e s could be removed by gentle means, we vortexed c e l l s w i t h a magnetic s t i r bar and analysed the m a t e r i a l s o l u b i l i z e d by t h i s process (surface p r o t e i n p r e p a r a t i o n ) . 87 F i g u r e 15. S D S - P A G E o f s u r f a c e p r o t e i n p r e p a r a t i o n s . Lane A , whole c e l l l y s a t e o f p a r e n t a l A.cryptum. Lane B , c e l l e n v e lope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane C, D , and E , s u r f a c e p r o t e i n p r e p a r a t i o n s o f p a r e n t a l A.cryptum, mutant-12, and mutant-15 r e s p e c t i v e l y . Lane A c o n t a i n s 0.3 g o f p r o t e i n . Lane B c o n t a i n s 0.2 / j g o f p r o t e i n . Lanes C, D , and E each c o n t a i n 0.05/^g o f p r o t e i n . 88 F i g u r e 16. Western b l o t o f a 12% SDS-PAGE d e v e l o p e d w i t h anti-A.cryptum a n t i b o d y . Lane A, whole c e l l l y s a t e o f p a r e n t a l A.cryptum. Lane B, c e l l e nvelope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane C, D, and E, s u r f a c e p r o t e i n p r e p a r a t i o n s o f p a r e n t a l A.cryptum, mutant-12, and mutant-15 r e s p e c t i v e l y . 89 Figure 15 i s a s i l v e r s t a i n e d SDS-PAGE comparing the m i g r a t i o n p r o f i l e of; p a r e n t a l whole c e l l l y s a t e (Lane A) , p a r e n t a l c e l l envelope p r o t e i n p r e p a r a t i o n (Lane B) and p a r e n t a l surface p r o t e i n p r e p a r a t i o n (Lane C) . The absence of the 31.6 kDa p r o t e i n i s i l l u s t r a t e d i n the surface p r o t e i n preparations of mutants-12 and -15, lanes D and E r e s p e c t i v e l y . Figure 16 i s a western immunoblot of Figure 15 and f u r t h e r suggests t h a t the 31.6 kDa p r o t e i n i s l o c a l i z e d at the c e l l surface of the p a r e n t a l s t r a i n and i s absent i n the mutant s t r a i n s . The v a r y i n g i n t e n s i t y of the 31.6 kDa p r o t e i n between preparations i n Figure 15 was d i s t u r b i n g . Though f i g u r e 16 shows s i m i l a r i n t e n s i t i e s of banding suggesting s i m i l a r antigen concentrations, a d i s t i n c t r e duction i n banding i n t e n s i t y i s observed i n f i g u r e 15. There are two p o s s i b l e explanations f o r t h i s discrepancy. The t i t e r of antibody s p e c i f i c to the 31.6 kDa p r o t e i n may be l i m i t e d i n the anti-A.cryptum a n t i s e r a and t h e r e f o r e would not r e f l e c t antigen c o n c e n t r a t i o n d i f f e r e n c e s during western immunoblot a n a l y s i s . An a l t e r n a t i v e e x p l a n a t i o n i s the presence of a s i m i l a r s i z e d p r o t e i n i n the whole c e l l l y s a t e w i t h was removed during the c e l l envelope p r o t e i n p r e p a r a t i o n . A monospecific p o l y c l o n a l antibody p r e p a r a t i o n was developed i n order to l o c a l i z e the 31.6 kDa p r o t e i n through immunogold l a b e l l i n g . A n t i s e r a t o the p a r e n t a l s t r a i n of A.cryptum was thoroughly adsorbed by the mutant-15 to generate the monospecific antibody p r e p a r a t i o n i l l u s t r a t e d i n Figure 17. Several 90 conclusions can be made from t h i s f i g u r e : The adsorption procedures generated antibody w i t h a s i n g l e s p e c i f i c i t y . Further evidence was provided t o suggest that s i m i l a r concentrations of the 31 .6 kDa antigen e x i s t i n whole c e l l l y s a t e s and c e l l envelope preparations of the p a r e n t a l s t r a i n ( F i g . 17, Lanes A & B) . The m o n o - s p e c i f i c i t y of the adsorbed antibody suggests that mutant-15 and the p a r e n t a l s t r a i n s d i f f e r only i n the 31 .6 kDa p r o t e i n . Mutant-15 and the p a r e n t a l s t r a i n were immunogold l a b e l l e d using the antibody p r e p a r a t i o n s p e c i f i c t o the 31 .6 kDa p r o t e i n . Figure 18 shows an even d i s t r i b u t i o n of gold beads i n d i c a t i n g t h a t the antigen was widely d i s t r i b u t e d over the surface of the p a r e n t a l s t r a i n . This was compared t o mutant-15 l a b e l l e d w i t h the monospecific antibody i n Figure 19 (a) , and the p a r e n t a l s t r a i n l a b e l l e d w i t h non-immune serum i n Figure 19 (b) . The i n t e n s i t y of gold bead l a b e l l i n g i n Figure 18 was not c o n s i s t e n t w i t h a l l p a r e n t a l c e l l s observed; some contained very few gold beads while others showed excessive l a b e l l i n g . This may be i n d i c a t i v e of phase v a r i a t i o n i n the p r o t e i n expression of the 31 .6 kDa p r o t e i n . 91 A B C D 9 7 . 4 -68-43--31.6 25.7-F i g u r e 17. Western b l o t o f a 12% SDS-PAGE d e v e l o p e d w i t h t h e m o n o s p e c i f i c a n t i - 3 1 . 6 kDa p r o t e i n p o l y c l o n a l a n t i b o d y p r e p a r a t i o n . Lane A, c e l l e n v e lope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane B, whole c e l l l y s a t e o f p a r e n t a l A. cryptum. Lane C , and D, c e l l e n v e l ope p r o t e i n p r e p a r a t i o n and whole c e l l l y s a t e o f mutant-15 r e s p e c t i v e l y . 9 2 V a r i a t i o n s i n the stringency of washing during immunogold l a b e l l i n g generated marked d i f f e r e n c e s i n the numbers of gol d beads l o c a t e d on the c e l l surface. Gentle c e l l resuspension using a pasteur p i p e t t e r e s u l t e d i n c o n s i s t e n t l a b e l l i n g as shown i n Figure 18. However, more vigorous washing i n a vortex mixer generated a l e s s evenly d i s t r i b u t e d p a t t e r n of gold beads as i l l u s t r a t e d i n Figure 20. In t h i s case, the 31.6 kDa p r o t e i n appeared to form aggregates on and away from the p a r e n t a l c e l l surfaces as i n d i c a t e d by the clumps of beads ( F i g . 20, arrow). The apparent loose a s s o c i a t i o n of the 31.6 kDa p r o t e i n w i t h the c e l l surface was a l s o shown i n Figure 21. In t h i s case the g o l d beads appeared to be deposited i n the shape of an A.cryptum c e l l suggesting that the 31.6 kDa p r o t e i n was adsorbed to the e l e c t r o n microscope g r i d and l e f t behind a f t e r the c e l l s were sheared away. Having e s t a b l i s h e d t h a t the 31.6 kDa p r o t e i n was a s s o c i a t e d w i t h the c e l l envelope, s o l u b i l i z a t i o n s t u d i e s were performed i n an attempt to p u r i f y the p r o t e i n . C e l l envelope p r o t e i n preparations were t r e a t e d w i t h detergents and d i f f e r e n t i a l l y c e n t r i f u g e d to separate the s o l u b i l i z e d p r o t e i n from the remaining envelope-bound p r o t e i n s . The s o l u b i l i z e d p r o t e i n s were subjected to western immunoblot a n a l y s i s . Only very f a i n t 93 F i g u r e 18. Immunogold bead l a b e l l i n g o f p a r e n t a l A.cryptum c e l l s w i t h a n t i - 3 1 . 6 kDa a n t i b o d y f o l l o w e d by u r a n y l a c e t a t e n e g a t i v e s t a i n i n g . Bar = 0.5 u i . 94 F i g u r e 19 (a) . Immune-gold bead l a b e l l i n g of mutant-15 wit h anti-31.6 kDa p o l y c l o n a l antibody followed by urany l acetate negative s t a i n i n g . Bar = 0.5 ^m. 95 Figure 19 (b) . Immune-gold bead l a b e l l i n g o f p a r e n t a l A.cryptum w i t h non-immune serum f o l l o w e d by u r a n y l a c e t a t e n e g a t i v e s t a i n i n g . Bar = 1 yum. 96 F i g u r e 20. Immunogold bead l a b e l l i n g of parental A.cryptum c e l l s with anti-31.6 kDa polyclonal antibody followed by uranyl acetate negative staining. C e l l s were mixed vigorously during the washing steps. The arrow indicates 31.6 kDa protein aggregates at the c e l l surface. Bar = 1 yum. 97 F i g u r e 21. Immune-gold bead l a b e l l i n g of a p a r e n t a l s t r a i n A.cryptum c e l l p r i n t w i t h anti-31.6 kDa p o l y c l o n a l antibody. Bar = 1 yum. 98 banding was observed through treatment w i t h guanidine HCl, urea or combined guanidine HCl-urea-EDTA and as such, was not considered an e f f e c t i v e approach to p u r i f i c a t i o n . I n h i b i t i o n assays were attempted using the monospecific antibody or the 31.6 kDa surface p r o t e i n p r e p a r a t i o n but the r e s u l t s were i n c o n c l u s i v e . A f t e r t r e a t i n g the c e l l s w i t h the monospecificantibody, the c e l l s formed l a r g e aggregates. T r e a t i n g the p l a t e s w i t h the surface p r o t e i n preparations r e s u l t e d i n high background readings and as such l i m i t e d the usefulness of t h i s technique. 99 DISCUSSION The purpose o f t h i s r e s e a r c h was t o e s t a b l i s h t e c h n i q u e s f o r s t u d y i n g a c i d o p h i l e adherence to m i n e r a l s u r f a c e s . T h i s i s the f i r s t s tudy on the adherence p r o p e r t i e s o f the a c i d o p h i l i c h e t e r o t r o p h , A.cryptum. The r e s u l t s c l e a r l y demonstrate t h a t t h i s organism b i n d s t o c h a l c o p y r i t e and p y r i t e . B i n d i n g was demonstrated by assays which measured c e l l p r o t e i n and r e a c t i v i t y w i t h a s p e c i f i c a n t i b o d y . The a c i d o p h i l i c h e t e r o t r o p h s are thought t o be impor tant e c o l o g i c a l p a r t n e r s i n T.ferrooxidans c o l o n i z a t i o n o f m i n e r a l s u r f a c e s . The h e t e r o t r o p h s consume o r g a n i c a c i d s and o t h e r m i c r o b i a l m e t a b o l i t e s which are t o x i c to the n u t r i t i o n a l l y f a s t i d i o u s a u t o t r o p h s . T h i s type o f in terdependence would b e n e f i t from a c l o s e a s s o c i a t i o n o f the organ i sms . The d a t a p r e s e n t e d here shows t h a t A.cryptum has the c a p a c i t y t o b i n d to s u l p h u r c o n t a i n i n g m i n e r a l s and t h e r e f o r e would be i n a p o s i t i o n to enhance the growth o f T.ferrooxidans. A concern when comparing the B i o - R a d assay and the Ore ELISA was the d i f f e r e n c e i n r e q u i r e d c e l l s n e c e s s a r y f o r s a t u r a t i o n . A 10 f o l d i n c r e a s e i n c e l l s added t o the Ore ELISA over c e l l s added t o the B i o - R a d assay was n e c e s s a r y t o r e a c h an e q u i v a l e n t s a t u r a t i o n p o i n t . That i s , w h i l e 60% o f the c e l l s added to the B i o - R a d assay had bound t o the c h a l c o p y r i t e , o n l y 6% o f the c e l l s added were bound i n the Ore E L I S A . T h i s d i f f e r e n c e i n r e s u l t s i s l i k e l y due t o the s u b s t a n t i a l r e d u c t i o n i n m i n e r a l s u r f a c e a r e a 100 a v a i l a b l e f o r c e l l adherence i n the Ore ELISA as compared to the. Bio-Rad assay. Approximately 18 mg of mineral ore i s bound to each ELISA w e l l , of which a p o r t i o n of t h i s , t h a t i s i n v o l v e d w i t h the ore-glue i n t e r f a c e , would not have been a v a i l a b l e f o r c e l l attachment. When converted t o numbers of c e l l s bound per m i l l i g r a m of ore, the values recorded between assays were s i m i l a r . However the Bio-Rad assay u s u a l l y measured a s l i g h t l y higher number of bound c e l l s than the Ore ELISA. This v a r i a t i o n can be ex p l a i n e d by the inherent d i f f e r e n c e s between the assays. The Bio-Rad assay separates bound c e l l s from unbound c e l l s through d i f f e r e n t i a l c e n t r i f u g a t i o n . I t i s l i k e l y t h a t during c e n t r i f u g a t i o n , some unbound c e l l s as w e l l as weakly bound c e l l s are removed from suspension w i t h the p e l l e t i n g ore p a r t i c l e s . The Ore ELISA, w i t h i t s s e v e r a l washing steps, has greater stringency and detects only t e n a c i o u s l y bound c e l l s . As a r e s u l t , the Bio-Rad assay w i l l record a higher number of c e l l s bound. The Ore ELISA and Bio-Rad assay generated very s i m i l a r r e s u l t s on the k i n e t i c s of A.cryptum adherence to c h a l c o p y r i t e . Both b i n d i n g isotherms suggest t h a t each c e l l adheres independently to form a c e l l monolayer. Adherence occurs r a p i d l y and i s complete by 20 t o 25 min which compares w e l l w i t h data f o r T. ferrooxidans (Myerson & K l i n e , 1983; Tuovinen et a l . , 1983). 101 Studies have shown that the r e l a t i o n s h i p between pH and c e l l adherence depends on the s u b s t r a t e . Tuovinen (1983) and D i S p i r i t o (1983) found t h a t adherence to g l a s s beads increased w i t h decreasing pH but adsorption to mineral surfaces was independent, of pH. The l a t t e r i s comparable to the Ore ELISA r e s u l t s which showed l i t t l e r e l a t i o n s h i p between c h a l c o p y r i t e b i n d i n g and pH. The Bio-Rad assay r e s u l t s compared more c l o s e l y t o the g l a s s bead adsorption. These r e s u l t s could be e x p l a i n e d by the f a c t that the mineral i s the only surface exposed i n the Ore ELISA where as the mineral and the polypropylene microfuge tube w a l l are exposed to the c e l l i n the Bio-Rad assay. Therefore, attachment to polypropylene l i k e attachment to g l a s s beads, may be pH dependant. The e f f e c t of surface a c t i v e agents on i n c r e a s i n g the b i n d i n g of A.cryptum t o c h a l c o p y r i t e was s i m i l a r to that reported f o r T.ferrooxidans (Duncan et a l . , 1964; Kingma et a l . , 1979; Starkey, 1956). An explanation f o r t h i s observation i s t h a t the s u r f a c t a n t reduces the dynamic surface t e n s i o n enabling A.cryptum t o be more r e a d i l y adsorbed to the ore surface. In these s t u d i e s both A.cryptum and T.ferrooxidans gave s i m i l a r r e s u l t s . P r i o r to t h i s report the use of p u r i f i e d p r o t e i n s ( i e . BSA, g e l a t i n or SPP) i n a c i d o p h i l e adherence i n h i b i t i o n s t u d i e s had not been i n v e s t i g a t e d . Both BSA and g e l a t i n s t r o n g l y i n h i b i t c e l l adherence to c h a l c o p y r i t e . These p r o t e i n s probably act by 102 b l o c k i n g b i n d i n g s i t e s on the mineral surface. This a l s o appeared to be a g e n e r a l i z e d phenomenon o c c u r r i n g i n both A.cryptum and T. ferrooxidans attachment to ore. This f i n d i n g could have a serio u s impact on i n d u s t r i a l b i o l e a c h i n g processes. That i s , as the age and d e n s i t y of a c u l t u r e i n c r e a s e s , the concen t r a t i o n of secreted p r o t e i n s or other macromolecules w i l l i n c r e a s e . Therefore, i r o n i c a l l y , the c r u c i a l act of adherence may be i n h i b i t e d by macromolecules r e l e a s e d from A.cryptum and T. ferrooxidans. The i n h i b i t o r y e f f e c t s of BSA on c e l l adherence to surfaces i s not unique t o A.cryptum. For example, Gibbons (1972) has shown tha t the adherence of s e v e r a l species of o r a l b a c t e r i a to hydroxyapatite beads i s i n h i b i t e d by BSA. The exact mechanism by which A.cryptum attaches t o c h a l c o p y r i t e i s s t i l l u n clear. A l e c t i n - l i k e i n t e r a c t i o n was not detected given the l i m i t e d number of sugars i n v e s t i g a t e d . Supplemental st u d i e s on polysaccharide mediated adherence w i l l v e r i f y t h i s f i n d i n g . Many p u t a t i v e i n h i b i t o r s enhanced c e l l adherence. The increased adherence recorded w i t h monovalent c a t i o n s i s l i k e l y due t o the reduced l a t e r a l r e p u l s i v e forces experienced by adsorbing p a r t i c l e s i n concentrated e l e c t r o l y t e s o l u t i o n s (Rutter and Vincent, 1984). Di v a l e n t c a t i o n s and c h e l a t i n g agents probably act i n the same way i n causing e l e v a t e d c e l l adherence to c h a l c o p y r i t e . Both chemical agents can mediate c e l l aggregation by l i n k i n g charged groups on two b a c t e r i a l c e l l s u rfaces. 1 0 3 I t doesn't appear as though c e l l v i a b i l i t y i s necessary f o r c h a l c o p y r i t e adherence since c e l l adherence t o c h a l c o p y r i t e was maintained i n the presence of b a c t e r i c i d a l agents. Many researchers are s t i l l d i v i d e d on t h i s question (Beck, 1967; Tuovinen et a l . , 1983). Reducing agents appear to have l i t t l e e f f e c t on c e l l adherence. The e f f e c t of sulphur b i n d i n g agents i s i n c o n c l u s i v e and should be viewed w i t h caution due to the i n s t a b i l i t y of these agents at such a low pH. Hydrophobic i n t e r a c t i o n s are considered t o be an e s s e n t i a l aspect i n b a c t e r i a l adherence to. surfaces and i n t e r f a c e s (Marshall & Cruickshank, 1973; Dahlback et a l . , 1981). Stenstrom (1989) drew a c o r r e l a t i o n between c e l l surface hydrophobicity of v a r i o u s Salmonella typhimurium s t r a i n s and adherence to the minerals; quartz, a l b i t e , f e l d s p a r , and magnetite. From t h i s study the r o l e of hydrophobicity i n the attachment of A.cryptum t o mineral surfaces i s not c l e a r . Enhanced adherence was observed under c o n d i t i o n s of high i o n i c c o n c e n t r a t i o n or low pH (high proton concentration) suggesting t h a t hydrophobicity may pla y a r o l e i n mineral adherence. However, t h i s c o n c l u s i o n was not supported by our c e l l surface hydrophobicity s t u d i e s . That i s , w hile some adherence-defective s t r a i n s of A.cryptum demonstrated increased c e l l surface hydrophobicity, other adherence-defective s t r a i n s showed increased h y d r o p h i l i c i t y . 104 D i r e c t comparison of attachment of d i f f e r e n t b a c t e r i a l species was achieved through competitive i n h i b i t i o n • s t u d i e s . I t was shown th a t A.cryptum and T. ferrooxidans do not compete f o r attachment to c h a l c o p y r i t e . The r e s u l t s suggest that each b a c t e r i a l species has unique attachment s i t e s on the c h a l c o p y r i t e mineral surface. This f i n d i n g i s of great s i g n i f i c a n c e i n the study of m i c r o b i a l adherence to mineral surfaces. I t provides the f i r s t documented evidence to suggest that b a c t e r i a l attachment to mineral surfaces i s a s p e c i f i c process t h a t i s unique among d i f f e r e n t s t r a i n s of b a c t e r i a . In a d d i t i o n , the lack of competitive b i n d i n g between A.cryptum and T.ferrooxidans t o c h a l c o p y r i t e i m p l i e s t h a t the mechanisms of attachment f o r each c e l l type may d i f f e r . From an e c o l o g i c a l stand p o i n t , non-competitive attachment to mineral surfaces would b e n e f i t both A.cryptum and T.ferrooxidans. This would a l l o w the establishment of a c l o s e p h y s i c a l a s s o c i a t i o n between the two organisms necessary f o r c r o s s -feeding. In a d d i t i o n , the establishment and p r o l i f e r a t i o n of e i t h e r organism would not be r e s t r i c t e d by attachment s i t e c o n s t r a i n t s due to b i n d i n g competition between s t r a i n s . Several l i n e s of evidence suggest that a 31.6 kDa p r o t e i n may be i n v o l v e d i n the adherence of A.cryptum to c h a l c o p y r i t e . The 31.6 kDa p r o t e i n does not appear to be a s s o c i a t e d w i t h the c e l l s as t y p i c a l f i m b r i a e - l i k e s t r u c t u r e s . That i s , v i s i b l e surface 105 processes were not evident i n negative s t a i n e d micrographs or immunogold l a b e l l e d micrographs. Heat treatment or treatment wi t h reducing agents p r i o r to e l e c t r o p h o r e s i s d i d not generate detectable subunits of t h i s . p r o t e i n . The 31.6 kDa p r o t e i n appears t o be a s s o c i a t e d w i t h the outer membrane and can be removed from the surface of c e l l s by p h y s i c a l means. In a d d i t i o n , t h i s p r o t e i n may be at l e a s t , i n p a r t , hydrophobic i n nature. This was presumed because the p a r t i a l l y p u r i f i e d p r e p a r a t i o n (SPP) of t h i s p r o t e i n was impossible t o s o l u b i l i z e i n d i s t i l l e d water, and immunogold l a b e l l i n g i n d i c a t e d t h a t the p r o t e i n formed aggregates when dis l o d g e d from the c e l l s u r f a c e . Conclusive evidence v e r i f y i n g t h a t the 31.6 kDa p r o t e i n mediates mineral surface adherence i s s t i l l l a c k i n g . However, s e v e r a l observations were made which would suggest t h a t t h i s p r o t e i n i s i n v o l v e d i n attachment: Figure 14 and the antibody adsorption st u d i e s show t h a t t h i s i s the only p r o t e i n missing i n adherence-d e f e c t i v e mutants. A mutant showing enhanced ore adherence contained the 31.6 kDa p r o t e i n . The " f o o t p r i n t " l e f t by an adherent c e l l i n Figure 21 suggests t h a t the p r o t e i n may be i n v o l v e d i n attachment. This observation i s s i m i l a r t o what was described by M a r s h a l l (1971) who showed th a t mechanical shearing of b a c t e r i a from surfaces produced a polymer f o o t p r i n t . This argument i s f u r t h e r supported by the f a c t t h a t the only e f f e c t i v e i n h i b i t o r of c h a l c o p y r i t e b i n d i n g by A.cryptum was p r o t e i n . Convincing evidence t o demonstrate t h a t the 31.6 kDa p r o t e i n i s 106 d i r e c t l y i n v o l v e d i n c e l l adherence would be through i n h i b i t i o n s t u d i e s u s i n g p u r i f i e d p r o t e i n or monospecific antibody t o the p u t a t i v e adhesin. Both of these experiments were attempted w i t h l i m i t e d success due t o complications a r i s i n g from the type assays used. Further research i n t h i s area would help t o e l u c i d a t e the r o l e of the 31.6 kDa p r o t e i n i n c e l l adherence. Attempts t o i d e n t i f y the s t r u c t u r a l a s s o c i a t i o n of t h i s p r o t e i n w i t h the c e l l surface through c e l l treatment s t u d i e s were i n c o n c l u s i v e . I f the 31.6 kDa p r o t e i n i s i n v o l v e d i n mineral attachment, p r o t e i n a s e K or p h y s i c a l l y t r e a t e d c e l l s should have d i s p l a y e d reduced c h a l c o p y r i t e adherence. The r e s u l t s recorded, however, are not uncommon since many c e l l surface p r o t e i n s are i n a c c e s s i b l e or r e s i s t a n t t o exogenous enzymes. In a d d i t i o n , p h y s i c a l treatment of the c e l l s i s not l i k e l y to remove a l l surface components and as such may not e f f e c t c e l l adherence. For example, treatments which removed the haemagglutonin from Streptococcus sanguis c e l l surfaces d i d not reduce the b i n d i n g a b i l i t y of the t r e a t e d c e l l s (McBride, pers. comm.). I t should be noted t h a t mechanisms other than the 31.6 kDa p r o t e i n may be i n v o l v e d i n ore adherence. The i n a b i l i t y t o generate non-adherent mutants a f t e r exhaustive enrichments, suggests t h a t the other adherence f a c t o r s , necessary f o r c e l l v i a b i l i t y , may be i n v o l v e d . The l i m i t a t i o n s of the Bio-Rad assay prompted the development of 107 the Ore ELISA. The Ore ELISA can provide adherence measurements of s p e c i f i c c e l l types such t h a t competition f o r b i n d i n g s i t e s between two c e l l types, can be performed. The mineral s u b s t r a t e f o r adherence i s immobilized to the ELISA p l a t e , t h e r e f o r e , i n t e r f e r e n c e by mineral f i n e s i s e l i m i n a t e d . This assay i s p a r t i c u l a r l y adapted t o c h a r a c t e r i z a t i o n s t u d i e s : The p o t e n t i a l f o r m u l t i p l e runs of a given t e s t c o n d i t i o n , provides s t a t i s t i c a l l y s i g n i f i c a n t and r e l i a b l e r e s u l t s . A v a r i e t y of p o t e n t i a l i n h i b i t o r s can be screened w i t h l i t t l e a f f e c t on assay f i d e l i t y . When preparing the p l a t e s , various mineral substrates can be s u b s t i t u t e d f o r comparative a n a l y s i s . In a d d i t i o n , t h i s assay i s a powerful t o o l f o r screening adherence-defective mutants. An added fea t u r e of t h i s assay i s tha t i f c o l o u r development exceeds the det e c t a b l e A 4 0 5 range, the p l a t e s can be emptied, new subs t r a t e added and inc u b a t i o n shortened u n t i l d e s i r a b l e A 4 0 5 values can be read. 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