The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a "helper" protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.

No abstract

No abstract

We examined the ability of a soil bacterium, Klebsiella planticola strain DSZ, to degrade the herbicide simazine (SZ). Strain DSZ is metabolically diverse and grows on a wide range of s-triazine and aromatic compounds. DSZ cells grown in liquid medium with SZ (in 10 mM ethanol) as carbon source mineralized 71.6+/-1.3% of 0.025 mM SZ with a yield of 4.6+/-0.3 microg cell dry weight mmol(-1) carbon. The metabolites produced by DSZ during SZ degradation included ammeline, cyanuric acid, N-formylurea and urea. We studied the physiological adaptations which allow strain DSZ to metabolize SZ. Using scanning electron microscopy, we detected DSZ cells covering the surfaces of SZ crystals when the herbicide was used at high concentrations (0.1 mM). The membrane order observed by FTIR spectroscopy showed membrane activity at low temperature (4 degrees C) to assimilate the herbicide. Membrane fatty acid analysis demonstrated that strain DSZ adapted to grow on SZ by increasing the degree of saturation of membrane lipid fatty acid; and the opposite effect was detected when both SZ and ethanol were used as carbon sources. This confirms the modulator effect of ethanol on membrane fluidity.

No abstract