Effects of culture conditions on enhancement of 2,4-dinitrotoluene degradation by Burkholderia engineered with the Vitreoscilla hemoglobin gene

Growth and degradation of 2,4-dinitrotoluene (2,4-DNT) were compared in liquid cultures in shake flasks for Burkholderia sp. strain DNT and strain DNT engineered to produce Vitreoscilla (bacterial) hemoglobin (strain YV1). Parameters varied included aeration rate, initial 2,4-DNT concentration (50 and 200 ppm), and concentration and type of cosubstrate (yeast extract, succinate, casamino acids, and tryptic soy broth). 2,4-DNT degradation increased with increasing cosubstrate concentration and was greater for strain YV1 than strain DNT under most conditions tested; the greatest advantages of YV1 (up to 3.5-fold) occurred under limited aeration. A third strain (YV1m), derived from YV1 by repeated growth on 2,4-DNT-containing medium, demonstrated increased 2,4-DNT degradation (up to 1.3-fold compared to YV1) at 200 ppm 2,4-DNT. The growth profiles of the three strains with respect to each other were in general similar to those of the degradation patterns of 2,4-DNT.     

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> improve the cell density and insecticidal crystal proteins yield

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb⁺ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb⁻ strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb⁺ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb⁻ strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.     

Microbial consortia that degrade 2,4-DNT by interspecies metabolism: isolation and characterisation

Two consortia, isolated by selective enrichment from a soil sample of anitroaromatic-contaminated site, degraded 2,4-DNT as their sole nitrogensource without accumulating one or more detectable intermediates. Thoughoriginating from the same sample, the optimised consortia had no commonmembers, indicating that selective enrichment resulted in different end points.Consortium 1 and consortium 2 contained four and six bacterial speciesrespectively, but both had two members that were able to collectivelydegrade 2,4-DNT. Variovorax paradoxus VM685 (consortium 1)and Pseudomonas sp. VM908 (consortium 2) initiate the catabolismof 2,4-DNT by an oxidation step, thereby releasing nitrite and forming4-methyl-5-nitrocatechol (4M5NC). Both strains contained a gene similarto the dntAa gene encoding 2,4-DNT dioxygenase. They subsequentlymetabolised 4M5NC to 2-hydroxy-5-methylquinone (2H5MQ) and nitrite,indicative of DntB or 4M5NC monooxygenase activity. A second consortiummember, Pseudomonas marginalis VM683 (consortium 1) and P.aeruginosa VM903, Sphingomonas sp. VM904, Stenotrophomonasmaltophilia VM905 or P. viridiflava VM907 (consortium 2), was foundto be indispensable for efficient growth of the consortia on 2,4-DNT and forefficient metabolisation of the intermediates 4M5NC and 2H5MQ. Knowledgeabout the interactions in this step of the degradation pathway is rather limited.In addition, both consortia can use 2,4-DNT as sole nitrogen and carbon source.A gene similar to the dntD gene of Burkholderia sp. strain DNT that catalyses ring fission was demonstrated by DNA hybridisation in the secondmember strains. To our knowledge, this is the first time that consortia are shownto be necessary for 2,4-DNT degradation.     

Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

Heterologous expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> haemoglobin in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.     

Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp. JS867 under oxygen limited conditions

Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 undervarying degrees of oxygen limitation was examined. Complete 2,4-DNT removalwas observed under oxygen excess with near stoichiometric release (83%) of nitrite.Average kinetic parameters were estimated based on a dual-Monod biokinetic modelwith 2,4-DNT and O₂ as growth limiting substrates. The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO₂ ^-. Under aerobic conditions, _max, K_sDNT, andK_iNO were 0.058(0.004) hr^-1, 3.3(±1.3) mg 2,4-DNT/L, and 1.2(±pm0.2) hr^-1, respectively. At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced. JS867 was able to use NO₂ ^- as a terminal electron acceptor whengrown on glucose or succinate under anaerobic conditions. However, during growthon 2,4-DNT and under O₂-limited conditions, JS867 did not use released nitrite as electron acceptor. The nearly constant molar ratios of DNT removed over NO₂ ^- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued. No evidence of nitroreduction was obtained under the examined oligotrophic conditions. JS867 displayed a high affinity for oxygen consumption with K_SO2 value of 0.285(±0.198) mg O₂/L. Our results indicate thatunder oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.     

An investigation of the peroxidase activity of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin

In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and l-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6–7. The VHb protein proved to be stable up to 80 °C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and l-dopa the activity was several orders of magnitude lower. The Michaelis constants, were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Malin Kvist and Ekaterina S. Ryabova contributed equally to this work.     

Biodegradation of 2,4-dichlorophenol by a <Emphasis Type="Italic">Bacillus</Emphasis> consortium

As part of our effort at establishing microbial consortia of relevance for the bioremediation of xenobiotics polluted environments in Mexico, we assessed the aerobic biodegradation of 2,4-dichlorophenol (2,4-DCP) by a consortium of four Bacillus species that were isolated from a polluted soil by enrichment using a mixture of chlorophenols. The bacterial consortium effectively biodegraded 2-chlorophenol, 3-chlorophenol and 2,4-dichlorophenol at degradation rates of between 1.7 and 6.7 μmoles l⁻¹ h⁻¹. In the presence of NH₄Cl or KNO₂ as nitrogen sources_, 2,4-DCP was variously degraded. Under both conditions, cell biomass attained highest values of 350 and 450 mg l⁻¹ respectively, while the amounts of 2,4-DCP metabolized in 21 days reached peak values of 2.1 and 2.5 mM representing between 70 and 85% degradation respectively. Chloride releases during the same period were highest at 4.7 mM and 5.3 mM in the presence of the two nitrogen sources. The presence of free-chloride in the culture medium had a significant impact on the catabolism of 2,4-dichlorophenol.     

Anaerobic degradation of 2,4-dichlorophenoxyacetic acid by <Emphasis Type="Italic">Thauera</Emphasis> sp. DKT

Thauera sp. strain DKT isolated from sediment utilized 2,4-dichlorophenoxyacetic acid (2,4D) and its relative compounds as sole carbon and energy sources under anaerobic conditions and used nitrate as an electron acceptor. The determination of 2,4D utilization at different concentrations showed that the utilization curve fitted well with the Edward model with the maximum degradation rate as 0.017?±?0.002 mM/day. The supplementation of cosubstrates (glucose, acetate, sucrose, humate and succinate) increased the degradation rates of all tested chemical substrates in both liquid and sediment slurry media. Thauera sp. strain DKT transformed 2,4D to 2,4-dichlorophenol (2,4DCP) through reductive side-chain removal then dechlorinated 2,4DCP to 2-chlorophenol (2CP), 4-chlorophenol (4CP) and phenol before complete degradation. The relative degradation rates by the isolate in liquid media were: phenol?>?2,4DCP?>?2CP?>?4CP?>?2,4D?≈?3CP. DKT augmentation in sediment slurry enhanced the degradation rates of 2,4D and chlorophenols. The anaerobic degradation rates in the slurry were significantly slower compared to the rates in liquid media.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Production of antithrombotic hirudin in <Emphasis Type="Italic">GAL1</Emphasis>-disrupted <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h^–1 produced a maximum hirudin concentration of 62.1 mg l^–1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.     

Fed-batch production of <Emphasis Type="SmallCaps">d</Emphasis>-ribose from sugar mixtures by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SPK1

d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l^–1 xylose and 20 g l^–1 glucose resulted in 4.78 g l^–1 dry cell mass, 23.0 g l^–1d-ribose concentration, and 0.72 g l^–1 h^–1 productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l^–1 xylose and 50 g l^–1 glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l^–1 and gave the best results of 46.6 g l^–1d-ribose concentration and 0.88 g l^–1 h^–1 productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.     

Early perception responses of<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> cells in response to lipopolysaccharides from<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis>

Lipopolysaccharides (LPS) are cell surface components of Gram-negative bacteria and, as microbe- / pathogen-associated molecular patterns, have diverse roles in plant–microbe interactions, e.g. LPS are able to promote plant disease tolerance through activation of induced or acquired resistance. However, little is known about the mechanisms of signal perception and transduction in response to elicitation by these bio-active lipoglycans. The present study focused on the involvement of LPS isolated from the outer cell wall of the Gram-negative bacterium Burkholderia cepacia (strain ASP B 2D) in the molecular mechanisms and components involved in signal perception and transduction and defense-associated responses in suspension-cultured tobacco (Nicotiana tabacum L.) cells. The purified LPS_B.cep. was found to trigger a rapid influx of Ca²⁺ into the cytoplasm of aequorin-transformed tobacco cells. An oxidative burst, concomitant with the production of reactive oxygen and nitrogen species was measured by chemiluminescence and fluorescence. These early perception responses were accompanied by K⁺/H⁺ exchange and alkalinization of the extracellular medium. Through the use of various inhibitors of the oxidative burst reaction, as well as scavengers of produced radicals, the biochemical basis of the cellular response to LPS_B.cep. elicitation was dissected, elucidated and compared to that induced by a yeast elicitor. These results suggest that LPS_B.cep. interacts with tobacco cells in a manner different from the response elicited by yeast elicitor.Abbreviations DDC Diethyldithiocarbamate - DMSO Dimethyl sulfoxide - DPI Diphenylene iodonium - H ₂ DCF-DA 2,7-Dihydrodichlorofluorescein-diacetate - LPS Lipopolysaccharides - NAC N-Acetyl-l-cysteine - PTIO 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - ROS Reactive oxygen species - YE Yeast elicitor     

High-frequency transformation of<Emphasis Type="Italic"> Lobelia erinus</Emphasis> L. by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated gene transfer

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a -glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3–4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high—45% per inoculated disc.Abbreviations BA: 6-Benzyladenine - 2,4-D: 2,4-Dichlorophenoxyacetic acid - GUS: -Glucuronidase - IAA: Indole-3-acetic acidCommunicated by G.C. Phillips     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P _alsSD promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic–anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P _alsSD promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.     

Novel Production of Isochainin by a Strain of <Emphasis Type="Italic">Streptomyces </Emphasis>sp<Emphasis Type="Italic">.</Emphasis> Isolated from Rhizosphere Soil of the Indigenous Moroccan Plant <Emphasis Type="Italic">Argania Spinosa</Emphasis> L

Summary An actinomycete strain (designated Ap1) isolated from the rhizosphere soil of Argania spinosa L. strongly inhibited the growth of two plant pathogens: Fusarium oxysporum f.sp. albedinis and Verticillium dahliae. The spore morphology suggested that the Ap1 strain belonged to the genus Streptomyces. The antifungal compound produced by Ap1 was purified by HPLC and identified as the polyene macrolide, isochainin, by NMR and mass spectroscopy. Ap1 showed normal biosynthesis of isochainin in comparison with S. cellulosae ATTC 12625, in which precursor-directed biosynthesis by feeding ethyl (Z)-16-phenylhexadec-9-enoate to the culture medium is required. In addition, Streptomyces sp. strain Ap1 produces isochainin with a 6.5-fold higher concentration than Streptomyces cellulosae ATTC 12625.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase