Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.

Microbial breakdown of halogenated aromatic pesticides and related compounds

Biodegradation of halogenated organic compounds.

Exposure of dry oxic paddy sol to submerged anoxic conditions resulted in a decrease of the soil redox potential and initiation of CH4 production. However, the most probable numbers (MPN) of hydrogenotrophic and acetotrophic methanogens did not change upon flooding the soil and upon initiation of CH4 production, and stayed constant at 105−106 and 104−105 g−1 d.w. soil, respectively. Cells of Methanobacterium strain Bab1 added to anoxic soil suspensions mostly survived drying and storage under an air atmosphere. Initiation of CH4 production was not affected by storage of dry soil under N2, air, or O2 atmosphere. However, it was negatively affected by storage under the air at high negatively affected by storage under air at high relative humidities, under moist conditions, or by using only the small (< 100 μ m) soil particle fraction. All these treatments also resulted in decreased contents of dissolved organic carbon (DOC). The soil's capacity for initiation of CH4 production could be regenerated either by addition of glucose or by exposure to H2 and addition of reducing agents. The establishment of decreased redox potentials and the provision of methanogenic substrates by heterotrophic bacteria, but not the number of methanogenic bacteria, seemed to be the essential factors for initiation of CH4 production after submergence of oxic paddy soil. CH4 production could also be initiated in forest and arable soils. Here, however, the population of methanogenic bacteria (MPN) increased from about 103 to 105−106 g−1 d.w. when methanogenesis started.

Factors influencing the population of methanogenic bacteria and the initiation of methane production upon flooding of paddy soil

Plasmids are extrachromosomal pieces of double-stranded circular DNA which have the capability to replicate independently of the host chromosome, yet coexist with it [1]. To date, many species of bacteria isolated from diverse habitats are known to contain plasmid DNA [2,3]. Some plasmids are stable and can be maintained through successive generations by being partioned to each daughter cell during cell division. This allows each cell to receive at least one plasmid copy.

In recent years, plasmids have been observed in a wide variety of bacteria. In part, this is due to the development of new procedures that allow the detection, isolation, and molecular characterization of plasmid DNA. When working with some plasmid-containing bacteria, it is often desirable to obtain a plasmid-cured derivative. This allows a direct comparison to be made between the plasmid-containing and plasmid-cured cells. Some plasmids undergo spontaneous segregation and deletion. However, the majority are extremely stable, and require the use of curing agents or other procedures (elevated growth temperature, thymine starvation), to increase the frequency of spontaneous segregation [4]. The usefulness of curing agents is unpredictable in many bacterial strains, as there are no standard protocols applicable to all plasmids. However, there are some procedures that have provided good results with certain species.

The aims of this manuscript are to summarize and review plasmid curing agents and procedures. In view of the importance of plasmids in specifying antibiotic and metal-resistance; metabolic properties; pathogenicity; host specificity and nodulation ( Rhizobium spp.); conjugal properties, and replication-maintenance properties [2], reproducible procedures for obtaining plasmid-cured derivatives are necessary.

Plasmid curing in bacteria

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.

Heinrich Keil

Papers

Degradation of 4-chlorobenzoate by Pseudomonas sp. CBS3: Induction of catabolic enzymes

Degradation of aromatic carboxylic acids by acinetobacter.