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The cell membrane of bacteria consists of a lipid bilayer with inserted proteins. The lipids provide a hydrophobic barrier to hold the aqueous cytoplasm in and keep the habitat molecules out. In general, polar molecules (carbohydrates) and charged molecules (ions, amino acids and side chains of larger molecules) do not pass through, except by very slow passive diffusion. Certain molecules do pass through the cell membrane because they are non-polar, hydrophobic or membrane soluble, such as glycerol and fatty acids, sterols, aromatic compounds and some amino acids (phenylalanine). Other molecules can pass through simply because they are small, such as water, short alcohols, ammonia, urea and dissolved gases (mostly O2 and CO2). The mechanisms of obtaining nutrient molecules and ions from the environment are regulated by the cell membrane chemistry and its proteins. These are diffusion, facilitated diffusion by binding to membrane receptors, and active transport through specific protein channels or transporters. The latter mechanism requires energy, such as an established electrochemical gradient across the membrane, or using high-energy bonds such as ATP dephosphorylation. The active transport mechanisms are more effective at translocation of nutrients into the cell, especially at low concentrations or against the concentration gradient. The proteins involved are under physiological regulation. They are translated from the genes into proteins only when they are required, then inserted into the cell membrane and activated. Only molecules that are small enough to pass through the membrane and its proteins can be used as nutrients. Therefore, cells are limited to monomers or short oligomers of amino acids, nucleic acids, saccharides, short lipids or other small molecules. Most species have several substrate transport mechanisms that allow the cell to use a variety of nutrients from the habitat, depending on what is available or missing outside. The ability and efficiency of each species or strain to import particular nutrients for growth are very useful in species identification.

Many genera of bacteria, particularly from certain taxa such as the Actinobacteria, release small molecules into their environment which prevent cell growth or cause lysis of susceptible cells. These secondary metabolites are known as antibiotics when they are effective at low concentrations. Antibiotics are usually produced when nutrients are rare, as in conditions when competition for nutrients is high. Secondary metabolites fall into three categories, that are derived from: (i) acetate-mal-onate condensation; (ii) condensation of carbohydrates or aminocyclitol; and (iii) oligopeptides, rarely long enough to be synthesized on ribo-somes. The resulting molecules tend to be membrane permeable and pass through the cell membrane. The mechanisms of function are very varied, and can interfere with one or more aspects of nutrient intake, anabolic or catabolic metabolism or synthesis of structural molecules. In Myxobacteria, where an estimated 60-80% of isolates produce antibiotics, they are bactericidal and cause lysis of susceptible bacteria. Other antibi otics target eukaryotic predators such as amoebae (Lancini and Demain, 1999). For example, Serratia marcescens (prodigiosin), Chromobacterium violaceum (violacein), Pseudomonas pyocyanea (pyocyanine) and P aerugi-nosa (phenazines) cause the encystment or lysis of amoebae that would ingest them. Other secondary metabolites are scavengers for cations (ionophores), or for iron (siderophores). These are sufficiently effective at removing specific ions or minerals from the habitat, especially where the ions are rare, so as to starve out species that do not have them. They are found in all strains of Nocardia, Streptomyces and Micromonosporea.

When bacteria are starved of nutrients, or in unfavourable conditions, they become physiologically inactive. Certain genera are able to produce spores, which are a differentiated state, with a resistant wall. In Actinobacteria, chains of spores form at the tips of growing filaments, in response to localized starvation. The pattern and shape of spores are important in species identification in the Actinobacteria. Other genera form a spore inside the parent cell, which is called an endospore. These include Bacillus, Clostridium, Sporosarcina and Thermoactinomyces. All species have preferences for optimum temperature, pH and nutrients. However, as the temperature, moisture, nutrient availability and other chemical changes in the habitat are continuously fluctuating, it is important for cells to tolerate a certain degree of fluctuation. It is important for living cells to adapt physiologically or protect themselves from threatening conditions, faster than it would take to cause death. Several mechanisms are available to bacteria, as indicated below.

To resist depletion of nutrients, cells can switch to higher affinity cell membrane transporters, increase the membrane surface area to cytoplasm ratio by decreasing the bulk cytoplasm, modify their metabolism, and divide at smaller cell sizes. These changes occur over several hours as they require changes in gene expression. On the same time scale, cells adapt to changes in temperature by modifying membrane lipid compositions. At higher temperatures, lipid membranes would be too fluid and more permeable, whereas they become too stiff at cooler temperatures. In order to maintain the fluidity of the membrane relatively constant, longer fatty acids and branched fatty acids are incorporated as the temperature rises, and shorter unsaturated fatty acids dominate membrane composition in colder temperatures. When the temperature rises above an optimal range for the species, heat-shock response proteins are synthesized to protect proteins from denaturing, while cells become inactivated.

In contrast, when soils freeze, ice formation outside the cell excludes some salts so that the osmotic pressure in the remaining water films increases, and mimics desiccation as water becomes less accessible to cells. Furthermore, ice crystals forming inside and outside the cytoplasm can break the cell. To protect themselves, most bacteria accumulate solutes which prevent ice crystal formation and freezing. Some species, such as Pseudomonas syringae, are able to insert a protein in the outer membrane that promotes more regular ice crystals to reduce the incidence of cell lysis. Lastly, cells can partially dehydrate and bind the remaining water to prevent ice formation in the cytoplasm.

The response to desiccation involves accumulating solutes that remain soluble at high concentration without interfering with enzyme reactions, and that are non-toxic to the cell (Potts, 1994). These are molecules that are metabolic end-points, i.e. molecules that are not metabolic intermediates and will not be required for other reactions. Commonly used molecules in bacteria are betaine, ectoine, l-proline and trehalose. Others are also found, such as glycerol, sucrose, d-gluci-tol, d-manitol, l-taurine and small peptides.

We will consider briefly several bacteria that are common in soils (Fig. 1.31). Anaerobic species tend to be rare in surface-aerated soils, but can be found in deeper soils, or in saturated fields such as rice paddies or riparian zones. Mycobacteria are Gram-positive non-motile aerobic species, that form irregular, slightly branched cells. They contain very hydrophobic wax-like mycolic acids in the cell membrane. This group includes pathogenic bacteria that cause leprosy and tuberculosis. The Myxobacteria are Gram negative, strictly aerobic, gliding species (Dawid, 2000). They form extensive spreading colonies. Many species secrete enzymes that lyse bacteria on which they feed, whilst the Polyangum and Cytophaga genera have cellulolytic enzymes. When nutrients are exhausted, cells aggregate into a mound (fruiting body) with characteristic shapes and differentiate into dispersal spores that are resistant to desiccation and adverse conditions. These fruiting bodies can be 0.1-0.5 mm and visible under the dissecting microscope (with the exception of Cytophaga which are missing the fruiting body stage). A large number of secondary metabolites are known from Myxobacteria, including bioactive molecules and coloured pigments derived from carotenoids. Other bacteria are able to hydrolyse and use cellulose, such as the well-studied Cellulomonas and the genus Erwinia. The Arthrobacter genus is common in soils and grows on humus, where readily soluble and metabolizable molecules have been exhausted. They grow slowly as cocci, or faster as branching semi-filaments. The Actinobacteria grow as filaments (or mycelia) and are found exclusively in soils. They are Gram positive and aerobic, but there are several anaerobic genera. They can be grown on agar media and identified based on their spore and sporangia structures. Their spores are all resistant to desiccation, but not to heat (except Thermoactinomyces vulgarum). The Streptomycetes (and the Myxobacterium Nannocystis excedens) produce geosmin which is the characteristic earthy odour of moist soil. As for the Arthrobacter, Actinobacteria grow on 'difficult' substrates that are not degraded by most species, such as cellulose and chitin. Most known antibiotics were first isolated from Actinobacteria. The polyphyletic Pseudomonads are aerobic Gram-negative bacteria which are able to grow on very diverse substances. They are

Fig. 1.31. Growth forms of various bacteria. (A) Inactive Bacillus rod-shaped cell with an endospore and protein crystal inclusion (e.g. Bacillus thuringiensus). (B) Coccus cell (e.g. Acinetobacter). (C) Mycobacterium growth forms. (D) Arthrobacter coccus, rod and branching cell growth. (E) Pseudomonas with apical flagella. (F) Spirillum with polar flagella tufts. (G) Proteus with lateral flagella. (H) Typical spirochete (Spirochaetae) with two spiral flagella between both membranes. (I) The same as (H) in cross-section. (J) Streptomyces (Actinobacteria) filamentous growth with aerospores. (K) Micromonosporea (Actinobacteria) filamentous growth with spores forming in the substrate. (L) Myxococcus (Myxobacteria) cells aggregated in a mound, releasing exospore cells. Scale bar (A-I) 1 ^m, (J-L) 10 ^m.

Fig. 1.31. Growth forms of various bacteria. (A) Inactive Bacillus rod-shaped cell with an endospore and protein crystal inclusion (e.g. Bacillus thuringiensus). (B) Coccus cell (e.g. Acinetobacter). (C) Mycobacterium growth forms. (D) Arthrobacter coccus, rod and branching cell growth. (E) Pseudomonas with apical flagella. (F) Spirillum with polar flagella tufts. (G) Proteus with lateral flagella. (H) Typical spirochete (Spirochaetae) with two spiral flagella between both membranes. (I) The same as (H) in cross-section. (J) Streptomyces (Actinobacteria) filamentous growth with aerospores. (K) Micromonosporea (Actinobacteria) filamentous growth with spores forming in the substrate. (L) Myxococcus (Myxobacteria) cells aggregated in a mound, releasing exospore cells. Scale bar (A-I) 1 ^m, (J-L) 10 ^m.

important in soils because some are able to utilize heterocyclic and aromatic molecules (such as lignins and its by-products). Similarly, the Clostridia are able to grow on a variety of nutrients, such as proteins, polysaccharides, purines and small soluble molecules. They are anaerobic endospore-forming bacteria which release malodorous metabolic byproducts, such as acetate, butyrate, butanol, acetone and many other molecules. More common in soils are genera of the endospore-forming aerobic rods (e.g. Bacillus) and Gram-negative facultative anaerobes such as Klebsiella and Aeromonas.

Bacteria are important symbionts of other organisms in decomposition food webs. Numerous anaerobic or anaero-tolerant species occur in the intestinal tract of animals. They are common in the intestinal tract of soil invertebrates (such as termites) where they are assumed to contribute to digestion, as intracellular or intranuclear symbionts of some protozoa, or as extracellular symbionts or commensals of ciliates and other protozoa. For example, the surface ridges of several Hypermastigea or the surface of the sand ciliate Kentrophoros are covered in symbiotic bacteria. There are also species-specific associations between some entomophagic nematodes (Heterorhabditidae and Steinernematidae) and bacteria of the genus Xenorhabdus. In this example, the bacteria are released from the nematode when it is ingested by an insect. The released bacteria produce an antibiotic in the insect, that kills the insect host and prevents colonization and growth in the insect of other bacterial species. The nematode then feeds and reproduces on the dead and decomposing insect. This interaction perpetuates a mutual interdependence between the two species.

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