The lacZ gene of Escherichia coli, coding for beta‐galactosidase, is a widely used reporter gene for gene expression studies in microbial and animal systems. To demonstrate that it is also a powerful reporter gene in plants, lacZ was fused to 5′ regulatory elements of several genes known to be functional in plant cells. By measuring LacZ activities in transgenic plants containing these gene constructs, we showed that the reporter is correctly monitoring the regulatory properties of the well‐characterized promoters fused to lacZ. beta‐Galactosidase was assayed directly in plant extracts when they contained high levels of LacZ or, when LacZ was expressed at low level, by separating the endogenous and LacZ activities electrophoretically and detecting the enzymes with a fluorogenic substrate. The most outstanding property of the marker is its amenability to histochemical detection. Due to its stability, LacZ can be fixed in the tissue with glutaraldehyde without loss of activity and detected with high resolution by using XGal. We could reveal expression patterns unnoticed earlier for many of the regulatory elements studied. The chlorophyll a/b binding protein gene, expressed at very high levels in green tissues, is also expressed at a low level in the vascular cylinder of the root. The Agrobacterium T‐DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and the TR2′ gene was shown to be root specific in the intact plant and stimulated by wounding in the leaf tissue. The TR1′ gene, fused to nptII, shows similar characteristics suggesting co‐regulation of this tightly linked dual promoter.

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P450. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.

The genus Ammoniphilus is proposed for aerobic endospore-forming Gramvariable rod-shaped bacteria, which are ammonium-dependent, obligately oxa lotrop h ic and haloa I ka I i to lerant, oxidase-and catal ase-pos i t ive, mesop h i I ic and motile b y peritrichous flagella. Cell wall contained two electron-dense layers. The external layer consists of a chain of electron-dense granules morphologically resembling the cellulosomes of CIostridium thermocellum. Two species are described, Ammoniphilus oxalaticus gen. nov., sp. nov. and Ammoniphilus oxalivorans gen. nov., sp. nov. The type strains of these species are strains RAOx-1 (= DSM 11538) and RAOx-FS (= DSM 11537), respectively. Ammoniphilus strains were isolated from the rhizosphere of sorrel (Rumex acetosa) and from decaying wood. The strains require a high concentration of ammonium ions and use oxalate as the sole organic source of carbon and energy for growth; no growth factors were required. Growth occurred a t pH 6-8-9-5. The optimum temperature and pH for growth were 28-30 "C and 80-8.5. All strains grew in a saturated solution of ammonium oxalate, and tolerated 3 O/ O NaCI. Whole-cell hydrolysates contain meso-diaminopimelic acid and glucose. The menaquinone of the strains was MK 7, and the major cellular fatty acids were 12-methyl tetradecanoic, cis-hexadec-9-enoic and hexadecanoic acids. The G+C content of the DNA was 4 5 4 6 mol O/ O for A. oxalaticus and 42 mol O/ O for A. oxalivorans. The almost complete 165 rDNA sequence of three strains of the two species of Ammoniphilus shows that the genus falls into the radiation of the Clostridium-Bacillus subphylum of Grampositive bacteria. The closest phylogenetic neighbour of Ammoniphilus is Oxalophagus oxalicus. The DNA-DNA hybridization value between strains RAOx-1 and RAOx-FS was 3997%.

Two different dehalogenation enzymes were found in cell extracts ofMycobacteriumfortuitum CG-2. The first enzyme was a halophenol para-hydroxylase, a membrane-associated monooxygenase that required molecular oxygen and catalyzed the para-hydroxylation and dehalogenation of chlorinated, fluorinated, and brominated phenols to the corresponding halogenated hydroquinones. The membrane preparation with this activity was inhibited by cytochrome P-450 inhibitors and also showed an increase in the A4,, caused by CO. The second enzyme hydroxylated and reductively dehalogenated tetrahalohydroquinones to 1,2,4-trihydroxybenzene. This halohydroquinone-dehalogenating enzyme was soluble, did not require oxygen, and was not inhibited by cytochrome P-450 inhibitors.Actinomycetes are a large and variable group of microorganisms occurring in many habitats, where they take part in the degradation of organic material. Many microbial strains degrading various aromatic pollutants have been isolated from this group. Enzymes so far characterized have been reported to be monooxygenases, hydroxylases, or dioxygenases (for a review, see reference 31). Actinomycetes, including those degrading xenobiotic compounds, have also been shown to be environmentally tenacious, making them a potential source of microorganisms for bioremediation (6,18,29,30).Mycobacterium fortuitum CG-2 was isolated from a tetrachloroguaiacol enrichment culture and shown to degrade several chlorinated phenols, guaiacols, and syringols at micromolar levels (15). Pentachlorophenol (PCP) was shown to be mineralized to CO2, and tetrachloro-para-hydroquinone (TeCH) was detected as an initial intermediate. We have shown that the degradation of polychlorophenols, guaiacols, and syringols proceeds through chlorinatedparahydroquinones in three different Rhodococcus chlorophenolicus strains (1,3,12,13,15) and is mediated by a membrane-associated cytochrome P-450 enzyme (28). The product of this para-hydroxylation, TeCH, is further orthohydroxylated by a soluble halohydroquinone dehalogenase (4,14,28).In this paper, we report on the enzymatic dehalogenation of chlorinated, brominated, and fluorinated phenols and hydroquinones by M. fortuitum CG-2 cell fractions. We show that the halogenated phenols were converted to halogenated para-hydroquinones by a membrane-associated cytochrome P-450 enzyme. The halohydroquinones formed by the para-hydroxylation were further dehalogenated by a soluble halohydroquinone dehalogenase. The results show that M. fortuitum CG-2 dehalogenases are similar to those previously described by us for R. chlorophenolicus PCP-1 and CP-2 (3, 4, 14, 15, 28). MATERIALS AND METHODSBacterial strains and culture conditions. M. fortuitum CG-2 (12, 16) was grown in nutrient broth-yeast extract medium (21) in a gyratory shaker at 28°C. Two-day-old cultures were harvested or induced by the addition of increasing amounts (10 to 50 FtM) of PCP at 24-h intervals and then harvested.Cell fractionation. Cells were harvested by centrifugation (3,000 x g, 10 min, 4°C), resus...

An aerobic mixed bacterial culture (CL-EMC-1) capable of utilizing methyl tert-butyl ether (MTBE) as the sole source of carbon and energy with a growth temperature range of 3 to 30 degrees C and optimum of 18 to 22 degrees C was enriched from activated sludge. Transient accumulation of tert-butanol (TBA) occurred during utilization of MTBE at temperatures from 3 degrees C to 14 degrees C, but TBA did not accumulate above 18 degrees C. The culture utilized MTBE at a concentration of up to 1.5 g l(-1) and TBA of up to 7 g l(-1). The culture grew on MTBE at a pH range of 5 to 9, with an optimum pH of 6.5 to 7.1. The specific growth rate of the CL-EMC-1 culture on 0.1 g l(-1) of MTBE at 22 degrees C and pH 7.1 was 0.012 h(-1), and the growth yield was 0.64 g (dry weight) g(-1). A new MTBE-utilizing bacterium, Variovorax paradoxus strain CL-8, isolated from the mixed culture utilized MTBE, TBA, 2-hydroxy isobutyrate, lactate, methacrylate, and acetate as sole sources of carbon and energy but not 2-propanol, acetone, methanol, formaldehyde, or formate. Two other isolates, Hyphomicrobium facilis strain CL-2 and Methylobacterium extorquens strain CL-4, isolated from the mixed culture were able to grow on C(1) compounds. The combined consortium could thus utilize all of the carbon of MTBE.