ITGA01 18/7/06 18:06 Page iii Introduction to Geomicrobiology Kurt Konhauser ITGC03 18/7/06 18:11 Page 93 3 Cell surface reactivity and metal sorption One of the consequences of being extremely 3.1 The cell envelope small is that most microorganisms cannot out swim their surrounding aqueous environment. Instead they are subject to viscous forces that 3.1.1 Bacterial cell walls cause them to drag around a thin film of bound water molecules at all times. The im- Bacterial surfaces are highly variable, but one plication of having a watery shell is that micro- common constituent amongst them is a unique organisms must rely on diffusional processes material called peptidoglycan, a polymer con- to extract essential solutes from their local sisting of a network of linear polysaccharide milieu and discard metabolic wastes. As a (or glycan) strands linked together by proteins result, there is a prime necessity for those cells (Schleifer and Kandler, 1972). The backbone to maintain a reactive hydrophilic interface. of the molecule is composed of two amine sugar To a large extent this is facilitated by having derivatives, N-acetylglucosamine and N-acetyl- outer surfaces with anionic organic ligands and muramic acid, that form an alternating, and high surface area:volume ratios that provide repeating, strand. Short peptide chains, with four a large contact area for chemical exchange. or five amino acids, are covalently bound to some Most microorganisms further enhance their of the N-acetylmuramic acid groups (Fig. 3.1). chances for survival by growing attached to They serve to enhance the stability of the submerged solids. There, they may adopt a entire structure by forming direct or interchain more hydrophobic nature to take advantage of cross-links between adjacent glycan strands. The the inorganic and organic molecules that pre- peptide chains are rich in carboxyl (COOH) ferentially accumulate. Accordingly, through- groups, with lesser amounts of amino (NH2) groups out a microorganism’s life, there is a constant (Beveridge and Murray, 1980). interplay with the external environment, in Despite the enormous variety of bacterial which the surface macromolecules are modified species, most can be classified into two broad in response to changing fluid compositions categories: Gram-positive and Gram-negative. and newly available colonizing surfaces. In this This terminology has its basis in the cell’s response chapter we focus on how cellular design can to the differential staining technique developed facilitate the accumulation of metals onto by Christian Gram in 1884. The Gram stain microbial surfaces, often in excess of mineral involves using four chemicals on dried smears of saturation states. We then examine how model- bacteria in the following sequence: crystal violet, ing their chemical reactivity can be applied to iodine, ethanol, and safranin. Bacteria that are the environmental issues of contaminant bio- able to retain the crystal violet–iodine complex, remediation and biorecovery of economically even after decolorization with ethanol are called valuable metals. Gram-positive. Those that lose their purple ITGC03 18/7/06 18:11 Page 94 94 CHAPTER 3 N-acetylmuramic acid (M) N-acetylglucosamine (G) CH2OH O 1 Glycosidic bond CH2OH 4 O O 2 Glycan CH OH 2 4 strand O O 3 CH2OH O O O NH O OH NH CO M M M M M O H C CH O NH CO 3 CH3 G G G G G CH C M M M M M OH NH CO 3 HCCH3 CO CH3 G G G G G C M M M M CH3 L-alanine L-alanine 1 4 D-glutamine D-glutamine 4 2 Peptide Peptide cross-linkages chain L-lysine L-lysine 4 4 D-alanine D-alanine 3 (M) (G) (M) Figure 3.1 Structure of peptidoglycan. It is composed of strands of repeating units of N-acetylglucosamine and N-acetylmuramic acid sugar derivatives. The sugars are connected by glycosidic bonds, but the overall resilience comes from the cross-linking of the glycan strands by peptide chains. coloration and are counterstained with safranin abundances, most bacteria are Gram-negative, to become red are Gram-negative. It is now while the Gram-positive cells are distinguished recognized that these staining characteristics on the universal phylogenetic tree as two sister highlight some fundamental differences in the phyla (the Firmicutes and the Actinobacteria), chemical and structural organization of the cell united by their common cell wall structure. wall (Beveridge and Davies, 1983). In both cell types, the crystal violet–iodine complex pene- (a) Gram-positive bacteria trates the cell wall and stains the cytoplasm. Then during the decolorization step, the ethanol A large proportion of the work conducted on solubilizes some of the membranous material. This the ultrastructure and metal binding properties is where the inherent differences lie. In Gram- of Gram-positive cells has been done using a positive cells, their thick peptidoglycan walls common soil constituent, Bacillus subtilis. Under become dehydrated by the alcohol, the pores the transmission electron microscope (TEM), a in the wall close, and the crystal violet–iodine technique that permits resolution of objects as complex is prevented from escaping. By contrast, small as a few nanometers, these species are Gram-negative cell walls have thin peptido- observed having a single wall layer averaging glycan walls that cannot retain the stain when 25–30 nm thick, which consists of 30–90% the membranes are dissolved. In terms of global peptidoglycan. The remaining materials are ITGC03 18/7/06 18:11 Page 95 CELL SURFACE REACTIVITY AND METAL SORPTION 95 secondary polymers that are covalently attached while the other half is embedded in the peptido- to the peptidoglycan (Fig. 3.2). For instance, when glycan matrix by penetrating through its interstices B. subtilis is grown in the presence of phosphate, (Doyle et al., 1975). Some teichoic acids are also its wall has essentially two chemical compon- bound to membrane lipids, and they are called ents of roughly equal proportion; peptidoglycan lipoteichoic acids. and teichoic acid (Beveridge, 1989a). Teichoic When growth of B. subtilis is limited by the acids are either glycerol- or ribitol-based poly- availability of phosphate, teichoic acid synthesis saccharides, with a terminal (H2PO3) phosphoryl ceases and it is totally replaced by teichuronic group and glucose or amino acid residues (Ward, acid, a polymer made up of alternating sequences 1981). A phosphodiester group links the teichoic of N-acetylgalactosamine and carboxyl-rich glu- acid chain to N-acetylmuramic acid of the curonic acid, but lacking phosphate. Variations peptidoglycan. Teichoic acids provide a distinct in the type and quantity of secondary polymer asymmetry in composition between the wall’s indicate that the wall composition, at least for inner and outer surfaces because half extends B. subtilis, may be a phenotypic expression of the perpendicularly outwards into the external milieu, environment (Ellwood and Tempest, 1972). Stated A Peptidoglycan Plasma membrane 50 nm B Wall-associated protein Teichoic acid Lipoteichoic Chemoreceptor acid Peptidoglycan (~30 nm thick) Plasma membrane (not to scale) Electron transport enzyme Electron transport enzyme Permease ATPase Figure 3.2 (A) A TEM image of a Bacillus subtilis cell wall (courtesy of Terry Beveridge). (B) Representation of the overall structure of a Gram-positive bacterium. ITGC03 18/7/06 18:11 Page 96 96 CHAPTER 3 simply, the bacterium has the means to adapt bound to the lipid A is the “core”, consisting of the its biochemistry to compensate for geochemical unique sugars 3-deoxy-D-mannooctulosonate (also changes in its environment, and as will be dis- known as KDO) and L-glycero-D-mannoheptose cussed below, this has implications for it retain- (or heptose), along with N-acetylglucosamine, ing high surface reactivity. galactose, and a number of other sugars whose exact combination varies between species. Chemically (b) Gram-negative bacteria the core contains carboxyl and cationic amino + groups (NH4 ) that are cross-linked, usually with Much as Bacillus subtilis is the archetypal Gram- carboxyl groups present in excess. The outermost positive bacterium, Escherichia coli has largely region of the LPS is the “O-antigen.” It is made become the model Gram-negative bacterium. up of repeating carbohydrate units that are inter- The walls of E. coli are structurally and chemic- spersed with uronic acids and/or organic phos- ally complex (Beveridge, 1989a). External to the phate groups, the latter comprising 75% of the total plasma membrane is a very thin (3 nm thick) phosphorous associated with the outer membrane, peptidoglycan layer that makes up a mere 10% while the remainder is in the phospholipid. of the cell wall. This, in turn, is overlain by The remaining fraction of the outer mem- another bilayered structure, the outer membrane, brane contains two major types of proteins. that serves as a barrier to the passage of many Lipoproteins are confined to the inner face of unwanted molecules from the external environ- the outer membrane, and they serve to anchor ment into the cell (Fig. 3.3). The narrow region the outer membrane to the peptidoglycan (Di separating the plasma and outer membranes, Rienzo et al., 1978). The other proteins are called the periplasm, contains a hydrated, gel-like porins. They puncture the bilayer and func- form of peptidoglycan. In E. coli it is 12–15 nm tion as small-diameter (up to a few nanometers), thick and occupies approximately 10–20% of water-filled channels that completely span the the total cell volume. Within the periplasm is the outer membrane and regulate the exchange of peptidoglycan layer itself, a number of dissolved low-molecular-weight hydrophilic solutes into components such as amino acids, sugars, vitamins and out of the periplasm along a concentration and ions, and various macromolecules that are gradient (Hancock, 1987).