With continually expanding industry and a global dependence on fossil fuel hydrocarbons as well as agrochemicals, there are few environments that are not in some way affected by a spectrum of organic pollutants. These organic pollutants include the aliphatic hydrocarbons (e.g., alkanes from oil spills and petrochemical industry activities), alicyclic hydrocarbons (e.g., the terpenoid plant products), aromatic hydrocarbons (e.g., single aromatics, such as the petrochemical solvents benzene and toluene, and polyaromatics, such as pyrene), the chlorinated hydrocarbons (e.g., chlorinated aliphatics such as chloroform), chlorinated aromatics (e.g., chlorobenzenes and chlorinated polyaromatics such as PCBs, DDT, and dioxins), and N-containing aromatics (e.g., TNT). Because of the increasing awareness of the adverse effects of these organoxenobiotic pollutants, the soil microorganisms and associated genes (largely carried on plasmids, which are extrachromosomal pieces of DNA) involved in their degradation are of great interest to soil microbiologists. This interest focuses on the pollutant biodegradation capacity of microorganisms (which refers to both the breadth of the pollutants that they degrade and the degradation rates) and also on bioremediation of contaminated environments (Crawford and Crawford, 1996).

Some of the organic pollutants entering the soil environment are toxic to bacteria and are recalcitrant to mineralization. The reasons for the latter are numerous and include key physicochemical characteristics such as the way in which the pollutant partitions into the soil matrix—relevant only if related to metabolic processes. For example, the more hydrophobic polyaromatic hydrocarbons (PAHs) and poly-chlorinated biphenyls (PCBs) tend to bind strongly to the soil solid phase and limit microbial/enzyme access. Another reason for recalcitrance is the large number of enzyme-regulated steps and the number of well-regulated genes required for mineralization. Furthermore, since many of the organic pollutants under consideration are synthetic, there are often no corresponding genes coding for proteins existing in extant soil bacteria that can immediately catalyze the degradation process.

The highly chlorinated organic pollutants have been studied extensively in terms of their degradation and the evolution of their degradative genes. Although degradation is generally slow, particularly for the more chlorinated compounds, gene clusters have clearly evolved for their degradation. Structural and regulatory genes have high sequence homology, even though they have evolved as plasmid-borne genes in different soil bacterial genera from across the world (Daubaras and Chakrabarty, 1992). This evolution of gene-regulated bacterial degradative pathways (usually involving four to eight genes in each case) enables complete mineralization of a range of chlorinated pollutants, including chlorobenzenes used in a number of industrial processes and the chlorophenoxyacetic acids associated with some of the hormonal herbicides used in agricultural weed control. The degradation of many of the chlorinated organic pollutants is somewhat complicated by the fact that the bacterial removal of chlorine from the molecule (dehalogenation) occurs most readily under anaerobic conditions, and so bioremediation of contaminated environments containing both chlorinated and nonchlorinated hydrocarbons can be a complicated process involving management of both anaerobic and aerobic conditions. Although the anaerobic dehalogenation of PCBs is relatively well understood, obtaining reliable and useful bacterial isolates is fraught with difficulties (May et al., 1992).

¥ White rot fungi - lignin peroxidases, laccases

- Bacteria, fungi, algae (O2) Cytochrome P-450 Monooxygenase

PAH-Quinones—► Ring split jq O-Glucoside

Non-enzymatic_Phenol^ O-Glucuronide re-arrangement O-Sulfate


O-Xyloside h2o

Epoxide hydrolase


Cis, cis

Bacteria, algae O2 Ortho ^ muconic acid

Dioxygenase -► Cis" - dehydrogenase -"►Catechol^'''fission yg r dihydrodio| y g Meta fission^fc Hydroxymuconic semialdehyde

FIGURE 5.10 Schematic of soil microbial metabolism of polyaromatic hydrocarbons.

The soil bacteria and associated genes involved in the degradation of PAHs have, like the PCBs, been extensively studied and exploited in both bioremediation and generating bacterial biosensors for detecting these complex organic pollutants. For the bacteria that aerobically degrade PAHs (e.g., naphthalene, acenaphthylene, fluo-rene, anthracene, phenanthrene, pyrene, benz(a)anthracene, benzo(a)pyrene), attack often involves oxidation of the rings by dioxygenases to form cis-dihydrodiols (Fig. 5.10). The bacteria involved in these oxidative reactions include species of Mycobacterium (Kelley et al., 1993) and Pseudomonas (Selifonov et al., 1993). The dihydrodiols are transformed further to diphenols, which are then cleaved by other dioxygenases. In many bacteria, this precedes conversion to salicylate and catechol (Sutherland et al., 1995). The latter is a rather toxic, relatively mobile intermediate and any bioremediation of PAHs must consider and manage issues such as this both because of possible environmental hazards and because the catechol may inhibit the bacteria being harnessed for the bioremediation itself!

differentiation, secondary metabolism, and antibiotic production

Differentiation is when there is a change in bacterial activities from those associated with vegetative growth. This phenomenon is particularly associated with cell starvation, during which intracellular components are often transported and resynthesized into new compounds through secondary (i.e., non-growth-linked) metabolism. The compounds formed can include antibiotics, pigments, and even agents such as melanin that are protective against enzymatic attack. Antibiotics are produced by the actinobacteria (e.g., Streptomyces and Actinomyces) and by Bacillus and Pseudomonas. Antibiotics are often powerful inhibitors of growth and metabolism of groups of other microorganisms, with varying degrees of specificity. Streptomycin, for example, produced by certain species of Streptomyces, strongly inhibits a wide range of both gram-positive and gram-negative bacteria. Cycloheximide, on the other hand, inhibits only eukaryotes and is used as a fungal inhibitor in the bacterial plate count.

Antibiotic production by soil bacteria, involving secondary metabolism, has been harnessed for decades for a wide range of medical applications. Although antibiotic production has long been linked with chemical defense, the factors determining antibiotic production in soil suggest that antibiosis occurs only when the supply of available carbon is high (Thomashow and Weller, 1991). These conditions are likely to be met in the rhizosphere, the zone around seeds (the spermosphere) and relatively fresh plant or animal residues.

Although a considerable number of antibiotic-producing bacteria have been identified, evidence that antibiosis is a significant chemical defense strategy in the rhizosphere tends to be indirect. For example, strains of fluorescent pseudomon-ads can demonstrate high specific rates of production of the antibiotic phenazine, which is strongly inhibitory against Gaeumannomyces graminis var tritici (Ggt), the causal agent of take-all in wheat (Brisbane and Rovira, 1988). When Tn5 mutants of such pseudomonads are introduced into the wheat rhizosphere, the removal of antibiotic production has been associated with reduced control of Ggt (Thomashow and Weller, 1991).

The key genes and associated enzymes involved in antibiotic synthesis have now been characterized in some cases (e.g., for phenazine, see Blankenfeldt et al. (2004)) and hence offer new and more powerful approaches to investigate these secondary metabolic, chemical defense strategies of bacteria in soil; by probing for RNA-based antibiotic biosynthesis genes and/or by use of stable isotope (13C) probing of the rhizobacterial nucleic acid pool, questions of both the activation and the significance of antibiotic-mediated chemical defense can now be resolved.

The final two sections of this chapter have considered xenobiotics in two forms—antibiotics and persistent organic pollutants such as PCBs and PAHs. Nakatsu et al. (1991) pointed out that these two forms of organoxenobiotics have a key role in presenting soil bacteria with a major selection pressure. The molecular bases for organoxenobiotic resistance and catabolism are generally located in soil bacteria, on extrachromosomal plasmid DNA, the maintenance of which requires this selection pressure.


The structure and physiology of prokaryotes have been studied for more than a century, and we know much about their taxonomic and metabolic diversity that is directly relevant to their central role and essential activities in soil ecosystems. While summarizing what we know, we have also indicated the vast amount of important information that remains to be discovered. Arguably the majority of phylogenetic groups and strains that are abundant and important for soil processes have yet to be isolated and characterized. The diversity of the prokaryotes is significantly greater than that of higher organisms, but the lack of clear species definition limits our ability to apply concepts linking, for example, diversity to important ecosystem properties, such as stability and resilience. Elucidation of mechanisms generating and controlling prokaryote diversity within the soil environment and understanding the two-way relationships between prokaryotes and the soil, with its spatial and temporal heterogeneity, represent an enormous and exciting challenge. It is essential that this challenge is undertaken in order to provide a basis for understanding, and potentially, predicting the impact of environmental change on prokaryote diversity. Increased understanding of the links between phylogenetic diversity and functional and physiological diversity is also essential to determine the consequences of changes in prokaryote diversity and community structure on terrestrial biogeo-chemical cycling processes.

references and general reading

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