Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCGCTCATAAC-3'), RP1 antisense (5'-TCAGATGTCGGTCAGGGC-3'), FP2 sense nested (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGACATC-3').     

Some properties of a soluble methane mono-oxygenase from Methylococcus capsulatus strain Bath

Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme.     

Roles of the methane monooxygenase reductase component in the regulation of catalysis

The reductase component (MMOR) of the soluble methane monooxygenase isolated from Methylosinus trichosporium OB3b catalyzes transfer of 2e- from NADH to the hydroxylase component (MMOH) where oxygen activation and substrate oxidation occur. It is shown here that MMOR can also exert regulatory effects on catalysis by binding to MMOH or to the binary complex of MMOH and component B (MMOB), another regulatory protein. MMOR alters the oxidation-reduction potentials of the dinuclear iron cluster at the active site of MMOH. Although little change is observed in the potential for the first electron transfer to the cluster (E(1)0' = 76 mV), the E(2)0' potential value for the second electron transfer is increased from 21 to 125 mV. This shift provides a larger driving force for electron transfer from MMOR and favors transfer of two rather than one electron as required by catalysis. Similar positive shifts in potential are observed even in the presence of MMOB which has been shown to cause a 132 mV negative shift in the midpoint potential of MMOH in the absence of MMOR. MMOR is also shown to decrease the rate of reaction between the fully reduced MMOH-MMOB and O2 approximately 20-fold at 4 degrees C. However, the time course of the key catalytic cycle intermediate that can react with substates, compound Q, is unaffected. This implies a compensating faster decay of one or more of the intermediates that occur between diferrous MMOH and compound Q in the reaction cycle, thereby limiting potential nonproductive autodecay of these intermediates. Accordingly, an increase in single turnover product yield is observed in the presence of MMOR. Interestingly, MMOR can cause the redox potential increases, changes in rates, and the increase in product yield when present at only 10% of the concentration of MMOH active sites. Substrate binding is shown to induce negligible changes in the redox potentials. Two alternative regulatory schemes are presented based on (i) thermodynamic coupling of component binding and redox changes or (ii) dynamic interconversion of two states of MMOH promoted by MMOR.     

Oxidation of trichloroethylene and dimethyl sulfide by a marine Methylomicrobium strain containing soluble methane monooxygenase

Sixteen marine methanotrophic bacteria were isolated and 14 marine methanotrophic mixed cultures were obtained. They were assayed for soluble methane monooxygenase (sMMO) by naphthalene oxidation and only one isolate (strain NI) was positive. Strain NI degraded trichloroehylene (TCE) more efficiently than other methanotrophic isolates containing no sMMO only under copper limiting conditions. Dimethyl sulfide (DMS), one of the radiatively important trace gases released from the sea, was transformed to dimethyl sulfoxide (DMSO) by methanotrophs and the efficiency for the transformation of DMS to DMSO was not as much affected by the presence of sMMO as that of TCE. The taxonomical properties of strain NI and phylogenetic analysis based on 16S rDNA genes indicated that strain NI was a type I methanotroph belonging to the genus Methylomicrobium, and closely related to Methylomicrobium pelagicum. The partial mmoX gene of strain NI was amplified by the primers common to three other mmoX genes and its 270 bp were sequenced. 77 residues out of the 89 amino acids derived from the sequences were common among the four mmoX genes.     

Networks of transcriptional regulation encoded in a grammatical model

In a prospective multicenter study 68 out of 158 patients with HIV infection and malignant lymphoma were assigned to a risk-adapted induction therapy using the following algorithm: High-risk patients fulfilled 2 of 3 criteria: T4 lymphocytes <50/microL; WHO activity index 3 or 4; pre-existing AIDS-defining opportunistic infection. Normal-risk patients received 4 to 6 cycles of CHOP chemotherapy; those that achieved complete remission (CR) received zidovudine (500 mg/d) and interferon-alpha maintenance therapy (5 million units three times a week) for one year. High-risk patients received low-dose CHOP or vincristine/prednisone chemotherapy. Supportive care was performed with pentamidine inhalation and G-CSF. Intrathecal (it) methotrexate was given for CNS prophylaxis. The median survival was 634 days for 38 patients of the normal-risk group and 129 days for 30 patients of the high-risk group. 18 high-risk patients treated with low-dose CHOP had better survival (156 days) than 12 patients treated with vincristine/prednisone (72 days p=0.044). 68% of the patients in the normal-risk group achieved complete remission. 5 out of 18 high-risk patients treated with low-dose CHOP achieved complete remission. Three normal-risk patients developed fatal opportunistic infections during chemotherapy. Immune parameters deteriorated after CHOP induction and partially recovered with maintenance treatment. We conclude that the normal-risk patients survived longer than reported in most published studies. Toxicity was low. Low-dose CHOP seems to be superior to vincristine/prednisone therapy in high-risk patients.     

Nature and regulation of pistil-expressed genes in tomato

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.