Aerobic and anaerobic degradation of furan-3-carboxylate by Paracoccus denitrificans strain MK33

An organism identified as Paracoccus denitrificans was isolated from an enrichment culture with furan-3-carboxylate as its sole source of carbon and energy. The organism degraded furan-3-carboxylate under aerobic and — in the presence of nitrate - under anaerobic conditions. The aerobic degradation was initiated by an oxygenase reaction to form probably 2-hydroxy-3-furoate. Under anaerobic conditions the first intermediate was 3-furoyl-CoA which was reduced by NADPH to probably 4,5-dihydro-furoyl-CoA. Succinic semialdehyde was an intermediate in both aerobic and anaerobic mechanism of furan-3-carboxylate.Abbreviations F-3-C furan-3-carboxylate - 3-FCoA 3-furoyl-CoA     

The existence of an alternative electron-transfer pathway to the periplasmic nitrite reductase (cytochrome cd 1) in Paracoccus denitrificans

Exposure of aerobically-grown wild-type cells of Paracoccus denitrificans to a decreased aeration caused parallel increases in both PMS/ascorbate and succinate-linked activities of nitrite reductase. By contrast, the expression of the succinate-linked activity was considerably delayed in an insertion mutant specifically lacking the periplasmic 15 kDa cytochrome c-550. In this case the observed activity followed very closely the content of a 40 kDa cytochrome c. A subcellular fraction enriched in a haemoprotein of a similar apparent molecular weight showed the activity of cytochrome c peroxidase and was able to restore the antimycin-sensitive electron transport from membrane vesicles to nitrite reductase. It is concluded that P. denitrificans possesses an alternative nitrite-reducing pathway involving the 40 kDa cytochrome c instead of cytochrome c-550. This pathway branches from the respiratory chain after the cytochrome bc ₁ segment.Abbreviations PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulphate     

Isolation and characterization of Streptomyces lividans mutants deficient in intraplasmid recombination

Summary Plasmid pIF132 containing two direct repeats of the mel (melanin) sequence was used to monitor intraplasmid recombination. Five mutants of Streptomyces lividans TK64 deficient in intraplasmid recombination were isolated. Four contained additional defects in aerial mycelium formation, pigmentation, and nutrient requirements; among these two showed extensive amplification of chromosomal sequences. Mutant JT46 had no pleiotropic defects but had the most severe blockage in recombination. Only one of the mutants was slightly more sensitive to UV and two were slightly more sensitive to mitomycin C. Plasmid pWCL1 (containing pIJ702, pUC12, and HBVsAg sequences; Lee et al. 1986) could not stably replicate in TK64 without spontaneous deletions. In contrast the mutant JT46 maintained the integrity of pWCL1 much more stably.     

Nitrate-dependent [Fe(II)EDTA]2- oxidation by Paracoccus ferrooxidans sp. nov., isolated from a denitrifying bioreactor

Enrichments with [Fe(II)EDTA]2- as electron donor and nitrate or nitrite as electron acceptor were established using an inoculum from a bioreactor performing denitrification. A nitrate-reducing, [Fe(II)EDTA]2- oxidizing strain was isolated and named strain BDN-1. The G + C content of strain BDN-1 was 67%, and the organism was closely affiliated to Paracoccus denitrificans, P. pantotrophus and P. versutus by 16S rRNA sequence comparison. Results from DNA-DNA hybridization, rep-PCR, and whole cell protein analysis gave congruent results confirming the genotypic and phenotypic differences between strain BDN-1 and the other species of Paracoccus. From these results, we considered strain BDN-1 as a novel species for which we propose the name Paracoccus ferrooxidans. Apart from [Fe(II)EDTA]2-, BDN-1 could also use thiosulfate and thiocyanate as inorganic electron donors. Nitrate, nitrite, N2O, [Fe(II)EDTA.NO]2- and oxygen could be used by strain BDN-1 as electron acceptors. Repeated transfer on a culture medium with bicarbonate as the sole carbon source confirmed that strain BDN-1 was a facultative autotroph. [Fe(II)EDTA]2- oxidation dependent denitrification was also performed by other Paracoccus species, that were closely affiliated to P. ferrooxidans.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twentyone of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.In memoriam of Professor R. Y. Stanier     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans.

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.     

Heterotrophic cultivation of Paracoccus denitrificans in a horizontal rotating tubular bioreactor

In this work, the heterotrophic cultivation of bacterium Paracoccus denitrificans has been studied in a horizontal rotating tubular bioreactor (HRTB). After development of a microbial biofilm on the inner surface of the HRTB, conditions for one-step removal of acetate and ammonium ion were created. The effect of bioreactor process parameters [medium inflow rate (F) and bioreactor rotation speed (n)] on the bioprocess dynamics in the HRTB was studied. Nitrite and nitrogen oxides (NO and N₂O) were detected as intermediates of ammonium ion degradation. The biofilm thickness and the nitrite concentration were gradually reduced with increase of bioreactor rotation speed when the medium inflow rate was in the range of 0.5–1.5 l h⁻¹. Further increase of inflow rate (2.0–2.5 l h⁻¹) did not have a significant effect on the biofilm thickness and nitrite concentration along the HRTB. Complete acetate consumption was observed when the inflow rate was in the range of 0.5–1.5 l h⁻¹ at all bioreactor rotation speeds. Significant pH gradient (cca 1 pH unit) along the HRTB was only observed at the highest inflow rate (2.5 l h⁻¹). The results have clearly shown that acetate and ammonium ion removal by P. denitificans can be successfully conducted in a HRTB as a one-step process.     

Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity

Pichia stipitis strain NRRL Y-11,543 was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) to improve xylanolytic activity. A total of 20,000 mutants were screened for xylanase overproduction by observing the clear zones around the colonies on remazol-briliant-blue-xylan (RBB-xylan)-containing agar. Of 94 mutants isolated 11 of them were found to have enhanced xylanase activity compared to the parental strain. The most active mutant NP54376 had superior properties to the wild type which included: double the enzyme activity of wild type, a shorter generation time of 2.22 h compared to 3.13 h when grown on xylan, and an enhanced growth and yield of xylanase when low levels of xylose were added to the medium. Zymogram analysis of the crude enzyme preparations from both NP54376 and the wild type by isoelectric focusing showed multiple bands ranging between pI 4.2 and 7.4. No significant difference was observed in the K _m and V _max values of the parental strain and NP54376. K _m and V _max values of xylanase for birchwood xylan were 4.2 mg ml⁻¹ and 0.08 μmol min⁻¹ mg⁻¹ of protein, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of valine-resistant mutants of Nicotiana plumbaginifolia

Summary Haploid mesophyll protoplasts of Nicotiana plumbaginifolia were mutagenized by UV-irradiation. Protoplast-derived colonies were then selected for valine resistance on a medium containing 5 or 10 mM valine. From the resistant calli, plants were regenerated. Resistance was inherited as a recessive Mendelian character in seven clones. Mutations conferring valine resistance were shown to be allelic. Protoplast-derived cells of L-valine-resistant plants were also resistant to L-threonine. Resistance to valine was based on a reduced valine uptake rate.     

Growth and physiology of potassium-limited chemostat cultures of Paracoccus denitrificans

In potassium-limited chemostat cultures of Paracoccus denitrificans the maximum specific growth rate (µ_max) was found to depend on the input potassium concentration: At 0.21mM µ_max was 0.10–0.11 h^-1; at 0.44 mM 0.15–0.16 h^-1 and at 0.66 mM 0.20–0.21 h^-1. The plots of the specific rates of oxygen-, succinate-and potassium consumption against gave straight lines. The intracellular potassium concentration was a linear function of and varied from 1% (0.13 M) at a value of 0.034 h^-1 to 2.2% (0.29 M) at =0.26 h^-1; the potassium concentration gradient and the potassium concentration in the culture fluid in the steady state were dependent on the input potassium concentration. The potassium concentration gradient varied from 8,900-1,200. At all values 20–25% of the total energy production was used for potassium transport. 350,100 and 30 ATP molecules were calculated to be required to maintain one potassium ion intracellular during 1 h at values of 0.034, 0.197 and 0.257 h^-1 respectively. It is concluded that the amount of circulation of potassium is dependent on the potassium concentration gradient or on the potassium concentration in the culture in the steady state. The dependency of µ_max on the input potassium concentration was explained by the assumption that at low input potassium concentrations the net uptake of potassium (influx-efflux) is not rapidly enough to maintain the high potassium gradient in the existing cells and to establish it in the newly formed cells. At high values and at high input potassium concentrations µ_max is limited by the specific rate of oxygen consumption, which was found to be 11–12 mmol O₂ g dry weight^-1 h^-1 at µ_max for potassium-, succinate-and sulphate-limited chemostat cultures.     

Isolation and characterization of new bacteriophages for Myxococcus xanthus

Bacteriophages for Myxococcus xanthus of similar morphology to phage Mx4 were isolated from cultures of a variety of myxobacterial species. Phages similar to Mx1 and Mx8 were obtained by infecting M. xanthus with one of the phages of the Mx4 group that had been treated with either UV light or a chemical mutagen.The DNA molecules from the phages were characterized by electron microscopy. One phage, Mx113, contains an unusual type of terminal redundancy revealed by examination of denatured and re-annealed DNA.Several of the phages of the Mx4 group and the other two new phages, Mx113 and Mx811, were found capable of transducing genetic markers in M. xanthus.One phage, Mx416, was characterized in more detail. It establishes true lysogens in M. xanthus; the phage plaques on both a non-motile mutant and also on a wild-type host although it is restricted in the latter.We dedicate this paper to Professor Dr. Hans Kühlwein in the year of his retirement and in recognition of his many contributions to the study of Myxobacteria     

Distribution and analysis of plasmids inStreptococcus thermophilus

Summary In a survey of 35 strains ofStreptococcus thermophilus, 13 strains were found to harbor plasmid DNA. Most of these strains contained plasmid species varying in size from 2.2 to 7.15 kilobases. Only three strains had more than one plasmid species. Each of the nine distinct types of plasmid DNAs identified had two or more unique recognition sites for restriction endonucleases. The characteristics of the indigenous cryptic plasmids ofS. thermophilus may allow their development as cloning vectors useful in the genetic engineering of this species and other streptococci that are important in food production     

Isolation and characterization of Schizosaccharomyces pombe mutants phenotypically similar to ras1-

Summary We isolated mutants of Schizosaccharomyces pombe which have deformed cell morphology, are deficient in conjugation and poor in sporulation. This phenotype is characteristic of the ras1 defective mutant previously identified. Tests of the mutants for allelism using cell fusion showed that they define five complementation groups, one of which is ras1 itself. The others are named ral1 through ral4 (ras like). Mutants in ral3 or ral4 conjugate at a very low frequency, while the others apparently do not conjugate at all. Plasmid clones complementing ral1, ral2 or ral3, which apparently carry the respective gene, were isolated from S. pombe genomic libraries. Multiple copies of either the ral2 or the ral3 gene could partially restore mating ability in ral1 ^–strains. Multiple copies of the ras1 gene could partially restore mating ability in ral1 ^–and ral2 ^–strains. These results suggest that the ral1, ral2 and ras1 genes may function in a common pathway in that order. The ral3 gene may influence this pathway. Analysis of these gene products will aid identification of factors which interact with Ras proteins.