Chapter 2: Literature Review
C. Microbe-Metal Interactions
1. Microbial uptake of metals
Due to the small size and consequently high surface area to volume ratio of bacteria, their surfaces provide a large area for contact with the surrounding environment (Haferburg and Kothe, 2007). Since bacteria probably lack highly specific metal uptake systems (Haferburg and Kothe, 2007), the negative net charge of the cell envelope of bacteria assists them in accumulating metal cations from the environment (Collins and Stotzky, 1992). Two important metal uptake systems that exist in bacteria are siderophore- and heme-mediated iron uptake (Cornelis et al., 2011). Siderophores are strong extracellular Fe (III) chelators that assist in the transport of Fe (III) into the bacterial cell, where it is reduced and Fe (II) is then released leaving behind the iron chelator which is left intact allowing recycling (Cornelis et al., 2011). Heme is an important source of iron for bacteria and is not found unbound due to its potential toxicity and hydrophobicity (Wyckoff et al., 2005). Bacteria obtain heme from the hosts that they colonize and it is first extracted from hemoproteins such as haemoglobin or hemopexin. Once inside the cytoplasm of the bacteria, the heme is broken down into biliverdin and CO via heme oxygenase or it can be de-ferrated, releasing Fe (II) and leaving the tetrapyrrole ring intact (Letoffe et al., 2009; Wandersman and Delepelaire, 2004). The metal absorption to the cell envelope is influenced by the cell envelope components such as phosphoryl groups of lipopolysaccharides, carboxylic groups of teichoic and teichuronic acids, or capsule forming extracellular polymers such as sheaths (Haferburg and Kothe, 2007). Metal accumulation can take place via two processes, passive attachment onto the bacterial cell or via the active uptake into the bacterial cell. The passive uptake of metals is normally the dominant mode of metal accumulation due to nutrient scarcity in many natural environments such as soils (Haferburg and Kothe, 2007). The active uptake process is usually slower, requires energy and is dependent on metal-specific transport systems (Gadd, 1988).
31 2. Microbial mechanisms providing metal resistance
Metal resistance seems to be more prevalent in environmental systems than in pure cultures (Sprocati et al., 2006). An important resistance mechanism of bacteria to metals is the use of efflux transporters, characterized by a high substrate affinity. The transporters keep the metal concentration in the cytosol low via the excretion of over concentrated or toxic metals (Nies, 2003; Haferburg and Kothe, 2007). Another survival strategy is the release of metal binding compounds such as siderophores into the external environment of the bacterial cell where metals are then chelated and blocked from entering the cell (Haferburg and Kothe, 2007). This is an important mechanism as membrane transport systems of the cell cannot differentiate between potentially toxic and non-toxic metals (Haferburg and Kothe, 2007). A metal resistance mechanism for bacteria found in soil habitats is a combination of biosolubilization and bioprecipitation (Haferburg and Kothe, 2007), which typically involves the excretion of organic compounds that solubilize metals such as oxalates (Gadd, 1999). Some bacteria develop internal inclusion bodies e.g., polyphosphate granules, which bind the metal cations in the cytosol if they enter the bacterial cell and cannot be excreted via the efflux transporters (Gonzalez and Jensen, 1998). Another metal resistance mechanism, is the sorption of metals by the cell membrane in combination with the cell wall, which also facilitates bioreduction (Haferburg and Kothe, 2007).
The four main metal resistance mechanisms employed by microbes are summarized in figure 3.
32 Figure 3 (Adapted from Haferburg and Kothe, 2007): Overview of the four microbial metal resistance mechanisms. (X) - Cell constituents interacting with metal cations, (M) - Metal cation.
3. Metal toxicity for microbes
The ionic form of a metal is its most active form. Properties used to predict the toxicity of a metal ion are related to the solubility, stability and electrochemical characteristics of the metal (Venugopal and Luckey, 1978). In biological systems, the toxicity of a metal ion is associated with the difference in binding of these ions to biological structures such as tissues, cells, organelles etc., the stability of the metal ligand bonds and the form of the metal ion in the target biological structure (Venugopal and Luckey, 1978). The biological activity of a dissolved metal is correlated to its free ion concentration and the electrochemical properties consist of the oxidation potential, ionization potential, electropositivity, electronegativity, electron affinity and the oxidation state of the metal (Walker et al., 2003). The oxidation potential of a metal couple is the tendency of a metal ion to undergo oxidation from a lower oxidation state to a higher
33 oxidation state (Walker et al., 2003). Whilst the ionization potential is the difference in energy between the ground state and the state of ionization and it gives an indication of the electron affinity or electronegativity of the metal ion (Walker et al., 2003). Electropositivity is defined as the ability of a metal ion to lose electrons whilst electronegativity is defined as the ability of a metal ion to gain electrons or similarly the power of an atom to attract electrons to itself from a ligand (Rossotti, 1960). Electron affinity of a metal is the energy released when atom and ion are in their lowest energy states.
Yatsimirskii (1994) described the oxidation state of a metal ion as the charge of the metal ion in a purely ionic model for the complex. Overall, the toxicity of a metal is a combination of the physical and chemical properties of the metal and the interaction between metals and their biological targets (Luckey and Venugopal, 1977). Fe (II) and Mn (II) are trace elements and generally have a low toxicity. The minimum inhibitory concentration (MIC) of Mn (II) in E. coli was determined as 20 mM (Mergeay et al., 1985). The toxic potential of manganese and most other metals is determined by their ability to form complex compounds (Nies, 1999). Fe (II) is rapidly oxidized under aerobic conditions to Fe (III), which has a very low solubility and under most circumstances it is generally not toxic to aerobic bacteria (Nies, 1999).