Methyl <Emphasis Type="Italic">tert</Emphasis>-butyl Ether and <Emphasis Type="Italic">tert</Emphasis>-butyl Alcohol Degradation by <Emphasis Type="Italic">Fusarium solani</Emphasis>

Fusarium solani degraded methyl tert-butyl ether (MTBE) and other oxygenated compounds from gasoline including tert-butyl alcohol (TBA). The maximum degradation rate of MTBE was 16 mg protein h and 46 mg/g protein h for TBA. The culture transformed 77% of the total carbon to ¹⁴CO₂. The estimated yield for MTBE was 0.18 g dry wt/g MTBE.     

Toxic effect of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether on growth of soil isolate <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> T1/1

The toxic and growth inhibiting effects of methyl tert-butyl ether (MTBE) on the hydrocarbon-degrading Pseudomonas veronii T1/1 strain (isolated from gasoline contaminated soil) were studied. In our experiments, the MIC of MTBE was found to be 60 mM and the EC₅₀ was 51.7 mM. In the concentration range 0–30 mM, MTBE did not significantly influence the growth parameters of this bacterium, but at concentrations over 30 mM MTBE exerted a significant growth inhibiting effect. In the presence of 70 mM MTBE, the specific growth rate dropped from 0.4731 to 0.1201 h⁻¹, while the length of the lag period increased from 5.41 to 17.01 h and the yield coefficient declined from 0.2652 to 0.0718 g g⁻¹. MTBE at 100 mM inhibited the growth of this strain completely. These findings may have important environmental implications, as high concentrations of MTBE could influence the efficiency of soil and groundwater bioremediation processes significantly.     

Protective effects of baicalein on <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide-induced hepatic toxicity in rat hepatocytes

Summary Baicalein (BAL), a main flavonoid constituent of Scutellaria radix, was studied for its inhibitory effects on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity and oxidative damage in primary cultures of rat hepatocytes. In a preliminary study, baicalein revealed effective antioxidant properties in a test of its capacity to quench the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Further investigations showed that baicalein, at the concentrations of 1, 5, and 10M, decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and the formation of malondialdehyde (MDA) induced by 30min of pretreatment with t-BHP (1.5mM) in primary cultures of rat hepatocytes. Baicalein also attenuated t-BHP-induced mitochondrial depolarization as determined by a retention test of rhodamine 123 and DNA repair synthesis as evidenced by unscheduled DNA synthesis (UDS). In addition, baicalein decreased the 8-hydroxy-2-deoxyguanosine (8-OH-dG) content which acts as a DNA damage marker. The sum of the results suggests that the protective effect of baicalein against the cytotoxicity and genotoxicity of hepatocytes induced by t-BHP is due to its ability to quench free radicals.     

<Emphasis Type="Italic">n</Emphasis>-Alkane assimilation and <Emphasis Type="Italic">tert</Emphasis>-butyl alcohol (TBA) oxidation capacity in <Emphasis Type="Italic">Mycobacterium austroafricanum</Emphasis> strains

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.     

A comparative spin trapping study of the interaction of hypobromous and hypochlorous acids with <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide

Hypochlorite (HOCl/OCl^?) and hypobromite (HOBr/OBr^?) are shown to react with tert-butyl hydroperoxide with close rate constants (10.8 and 8.9 M^?1 s^?1, respectively). Using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone, both reactions are shown to proceed through decomposition of the hydroperoxide yielding butylperoxyl [˙OOC(CH₃)₃] and butoxyl [˙OC(CH₃)₃] radicals in a ratio depending on the hydroperoxide concentration. Thus, like hypochlorite, hypobromite can generate free radicals in reactions with organic hydroperoxides, which can be important for intensification of free-radical processes, e.g., lipid peroxidation at the chain branching stage.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether using bacterial strains

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen®. The biochemical oxygen demand of the cultures was measured using OxiTop®, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pathway,inhibition and regulation of methyl <Emphasis Type="Italic">tertiary</Emphasis> butyl ether oxidation in a filamentous fungus, <Emphasis Type="Italic">Graphium</Emphasis> sp.

The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority (∼95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.     

The Madagascan genus <Emphasis Type="Italic">Vaughania</Emphasis> is reduced to synonymy under <Emphasis Type="Italic">Indigofera</Emphasis> (<Emphasis Type="Italic">Leguminosae-Papilionoideae-Indigofereae</Emphasis>)

Summary  Eleven species comprising the Madagascan genus Vaughania are subsumed within the large pantropical genus Indigofera. Six new combinations are made; the remaining species were originally described in Indigofera.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan