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Archives of Microbiology (v.181, #6)

Purification and characterization of 2,6-dihydroxybenzoate decarboxylase reversibly catalyzing nonoxidative decarboxylation by Toyokazu Yoshida; Yutaka Hayakawa; Tsuyoshi Matsui; Toru Nagasawa (pp. 391-397).

A nonoxidative decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM12048. The enzyme activity was induced specifically by 2,6-dihydroxybenzoate. The purified enzyme was a homotetramer of identical 38 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate without requiring any cofactors. In the presence of KHCO₃, the enzyme also catalyzed the regioselective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate at a molar conversion ratio of 30%.

Keywords: 2,6-Dihydroxybenzoate decarboxylase; 2,6-Dihydroxybenzoate; Decarboxylation; Carboxylation; Agrobacterium tumefaciens IAM12048

Interaction between uranium and the cytochrome c ₃ of Desulfovibrio desulfuricans strain G20 by Rayford B. Payne; Laurence Casalot; Tessa Rivere; Jeffrey H. Terry; Lise Larsen; Barbara J. Giles; Judy D. Wall (pp. 398-406).

Cytochrome c ₃ of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c ₃ in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c ₃ (cycA) to lacZ. Instead, cytochrome c ₃ protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c ₃ with U(IV) was interpreted to be non-specific, since pure cytochrome c ₃ adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe₂O₃), and commercially available U(IV) oxide.

Keywords: Cytochrome c ; Desulfovibrio ; Uranium reduction

Degradation of o-xylene and m-xylene by a novel sulfate-reducer belonging to the genus Desulfotomaculum by Barbara Morasch; Bernhard Schink; Christoph C. Tebbe; Rainer U. Meckenstock (pp. 407-417).

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.

Keywords: Sulfate reduction; BTEX; Benzylsuccinate synthase; Desulfotomaculum

Use of TnKPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants by Gertrud Wiedemann; Peter Müller (pp. 418-427).

Transposon TnKPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination. The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans. Selected TnKPK2 insertions were introduced into the B. japonicum wild-type strain. The resulting mutants were subsequently tested for their symbiotic interactions with soybeans. As in Sinorhizobium meliloti, a B. japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules. The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained. Another gene was found to be highly similar to bacterial aspartases and thus was named aspA. The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237. The symbiotic phenotype of a B. japonicum aspA∷TnKPK2 mutant consisted of enlarged symbiosomes that made the system ineffective. In general, TnKPK2 is a suitable means for fast sequencing. In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants.

Keywords: In vitro transposon mutagenesis; Nodule development; Symbiotic interaction

First bacterial chalcone isomerase isolated from Eubacterium ramulus by Claudia Herles; Annett Braune; Michael Blaut (pp. 428-434).

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.

Keywords: Chalcone isomerase; Eubacterium ramulus ; Flavanone degradation; Naringenin; Naringenin chalcone; Isoliquiritigenin; Butein

Phylogenetic analysis of Verticillium species based on nuclear and mitochondrial sequences by Jan Fahleson; Qiong Hu; Christina Dixelius (pp. 435-442).

Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene (cob), the mitochondrial small subunit rRNA gene (rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.

Keywords: Mitochondrial and nuclear sequences; Partition-homogeneity test; Phylogeny; Verticillium species

Utilization of creatinine as an alternative nitrogen source in Corynebacterium glutamicum by Anne K. Bendt; Gabriele Beckers; Maike Silberbach; Anja Wittmann; Andreas Burkovski (pp. 443-450).

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, the use of creatinine as an alternative nitrogen source in Corynebacterium glutamicum, the identification of a membrane protein involved in creatinine uptake, the transcriptional regulation of the corresponding gene, and expression regulation of the gene encoding the creatinine deaminase are reported. As shown by mutant analyses, RNA hybridization experiments and real-time PCR, the expression of two genes, crnT and codA, is increased in response to nitrogen limitation, and regulation depends on the global nitrogen regulator AmtR. In addition, synthesis of creatinine deaminase during nitrogen starvation was shown by two-dimensional gel electrophoresis and MALDI-TOF-MS followed by peptide mass fingerprint analysis.

Keywords: Corynebacterium ; Creatine; Creatinine deaminase; Creatinine uptake; Nitrogen control; Nitrogen regulation

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Archives of Microbiology (v.181, #6)

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Archives of Microbiology (v.181, #6)