A directed approach to the selection of bacteria with enhanced catabolic activity.

AIMS: This study was aimed at selecting catabolicly-improved bacteria by in vitro evolution using a specially designed fermentor system. METHODS AND RESULTS: To facilitate this objective, genetic variation was induced by ultraviolet irradiation, and a selective pressure was subsequently exerted by gradual increases in the concentration of organic toxins. During a pilot experiment, a culture was forced to tolerate and catabolize a mixture of phenol and formaldehyde. The population developed a high resistance against formaldehyde and the specific degradation rate increased rapidly. Biochemical analysis of the mutants revealed an increase in the expression of enzymes involved in the pathway oxidizing formaldehyde. CONCLUSIONS: The fermentor system described is, in general, suitable for the selection of bacteria with enhanced catabolic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure is an alternative to conventional genetic engineering, providing efficient and genetically stable strains suitable for applications in the field of environmental biotechnology.

Phenol degradation by an enterobacterium: a Klebsiella strain carries a TOL-like plasmid and a gene encoding a novel phenol hydroxylase.

Although phenol catabolism is described for many different microorganisms, there is no example for such a pathway in an enterobacterial strain. Here we characterize a Klebsiella oxytoca strain that grows on phenol as the only source of carbon and energy. As the key enzyme of phenol degradation, phenol hydroxylase was purified to apparent homogeneity. Compared with other phenol hydroxylases, the Klebsiella enzyme differs with respect to several properties: (i) SDS-PAGE and gel-filtration analysis of the purified protein revealed that the enzyme is a monomer with a molecular mass of 156 kDa; (ii) steady-state kinetic measurements resulted in a K(m) value of 0.22 mM for phenol; and (iii) the enzyme is both dependent on NADPH/FAD and sensitive to EDTA. Further degradation of catechol, the reaction product of phenol hydroxylase, may occur via the effective meta-fission pathway often located on TOL or TOL-like plasmids. Such a plasmid was prepared from the Klebsiella strain and further characterized. The given data demonstrate that the isolated strain exhibits all characteristics of an efficient phenol-degrading microorganism.

Formation of hydride-Meisenheimer complexes of picric acid (2,4, 6-trinitrophenol) and 2,4-dinitrophenol during mineralization of picric acid by Nocardioides sp. strain CB 22-2.

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol. h(-1). g (dry weight) of cells(-1) in continuous cultures and 920 micromol. h(-1). g (dry weight) of cells(-1) in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a Nocardioides sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 +/- 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H-]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H--DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H--DNP formation indeed is a physiologically relevant step in picric acid metabolism.