UV and Ethyl Methanesulfonate Effects in Hyperthermophilic Archaea and Isolation of Auxotrophic Mutants of Pyrococcus Strains

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi. Received: 2 May 1996 / Accepted: 3 July 1996     

Interaction of <i>Acaena elongata</i> L. with Arbuscular Mycorrhizal Fungi under Phosphorus Limitation Conditions in a Temperate Forest

The aim of this study was to analyze the performance of Acaena elongata colonized by arbuscular mycorrhizal fungi (AMF) to different phosphorus (P) concentrations, as a measure of AMF dependency. A. elongata, is a species from soils where P availability is limited, such as temperate forests. Our research questions were: 1) How do different P concentrations affect the AMF association in Acaena elongata, and 2) How does the AMF association influence A. elongata’s growth under different P concentrations? A. elongata’s growth, P content in plant tissue, AMF colonization and dependency were measured under four P concentrations: control (0 g P kg⁻¹ ), low (0.05 g P kg⁻¹ ), intermediate (0.2 g P kg⁻¹ ) and high (2 g P kg⁻¹ ) in different harvests. A complete randomized block design was applied. A. elongata’s growth was higher under -AMF in intermediate and high P concentrations, and the lowest growth corresponded to +AMF in the low and intermediate P concentration. We observed a negative effect on the root biomass under +AMF in intermediate P concentration, while the P concentration had a positive effect on the leaf area ratio. The AMF colonization in A. elongata decreased in the highest P concentration and it was favored under intermediate P concentration; while the low and the high concentrations generated a cost-benefit imbalance. Our results suggest that the performance of some plant species in soils with low P availability may not be favored by their association with AMF, but a synergy between AMF and intermediate P concentrations might drive A. elongata’s growth.     

Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacteria: Isolation of auxotrophic mutants ofHaloferax mediterranei andHaloferax gibbonsii

The lethality and mutagenicity in ethyl methanesulfonate (EMS)-treated cells of five archaebacterial strains belonging to each of the three described genera of non-alkaliphilic halobacteria were investigated. In order to test the efficiency of the mutagenesis under a variety of experimental conditions, we chose the fast-growing halobacteriumHaloferax mediterranei as a model strain. A strong induced mutagenicity was found, since the spontaneous mutation rate (expressed as the rate of resistance to the antibiotic josamycin) increased up to 500-fold after mutagen exposure. The mutagenesis was also successfully used in obtaining auxotrophic mutants. Although a heterogeneous response to the induced effects caused after EMS exposure was detected for the other halophilic archaebacteria tested, a clear, efficient mutagenicity ofHalobacterium halobium andHaloferax gibbonsii was observed; auxotrophic mutants of this halobacterium were also produced. Optimal experimental conditions for EMS mutagenesis of some halobacteria were determined.     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI , active in mutation-specific reversion, and uvsC , a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli. Received: 23 September 1996 / Accepted: 2 December 1996     

The response of three chydorid species to temperature, pH and food

The responses of three chydorid species, Chydorus sphaericus(O.F. Müller), Alona affinis (Leydig) and Alonopsis elongata (Sars) to temperature, pH and food type were examined. Egg development time of all species decreased with increasing temperature, although the degree of change was different for each species. C. sphaericus had the fastest development time at all temperatures, and A. elongata the slowest. pH also affected the egg development time of each species differently. A. elongata failed to reproduce at low and medium pH, the egg development time of C. sphaericus was fastest at high pH while that of A. affinis was fastest at low pH. Food type was found to have significant effects on the population growth of individual species. C. sphaericus populations grew equally well in all three food types provided, and grew more than the other two species when fed on an algae culture, and filtered pond water. A. affinisand A. elongata populations grew best when fed on a detritus food source. The responses of each species to the different variables tested are discussed in relation to field observations of their distribution and abundance.     

Biodiversity and trophic structure of invertebrate assemblages associated with understorey red algae in a Laminaria digitata bed

Although the composition of invertebrate assemblages associated with kelps has motivated several studies in the recent past, little is known about assemblages associated with smaller, understorey macroalgae in these ecosystems. Here, the composition of invertebrate assemblages living within understorey macroalgae of a kelp (Laminaria digitata) forest has been studied over two sampling dates, and the structure of the food web investigated using stable isotopes. A total of 145 species of mobile fauna, mainly amphipods and gastropods, were identified. Although differences were date-dependent, we showed that the three species considered (Palmaria palmata, Mastocarpus stellatus, Ellisolandia elongata) sheltered different associated assemblages, including high host-specificity, which suggests that the animal diversity associated with rocky shores is enhanced by a high algal diversity. Overall, a dominance of gastropods was observed for the two-dimensional, leaf-like P. palmata, while the three-dimensionally structured species (M. stellatus, E. elongata) were characterized by a dominance of amphipods. Stable isotopes highlighted different trophic structures; E. elongata-associated assemblages were most likely relying on a dominant food source, sediment organic matter, while other assemblages were based on a wider diversity of food sources (algae, sediment, suspended organic matter). Our results illustrate the need to consider the microhabitats formed by understorey macroalgae in order to thoroughly assess the diversity and understand the functioning of coastal rocky ecosystems.     

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H₂-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate. Received: 17 September 1996 / Accepted: 5 November 1996     

New and noteworthyHabenaria spp. (Orchidaceae) from New Guinea and adjacent islands

5 new taxa ofHabenaria, namelyH. bougainvillae, H. elongata R. Br. var.leptophylla, H. ensigera, H. rechingeri andH. trichoglossa, are described and illustrated, with reference to affinities to related Australian and Indo-malayan species. The occurrence in New Guinea of severalHabenaria spp. typical for a savanna-like vegetation, led to look more thoroughly at these taxa:H. elongata R. Br. andH. ochroleuca R. Br., considered so far to be endemic in Northern Australia, andH. khasiana Hook. f., hitherto only known from southeastern Asia.Studies in the subtribeHabenariinae Bentham (Orchidaceae), 2.—Part 1: Candollea34, 357 (1979).Dedicated to Hofrat Prof. DrKarl Heinz Rechinger on the occasion of his 80th birthday. — On April 30, in the year 1927, the author and his brotherOtto Renz metKarl Heinz Rechinger on a small steam-boat in a stormy Aegaen Sea, travelling from Piraeus to the Northern Sporades Islands:Karl Heinz with destination to Chelidromia, the author andO. Renz to Skopelos. By this lucky chance a lasting friendship began.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996     

Creating auxotrophic mutants in Methylophilus methylotrophus AS1 by combining electroporation and chemical mutagenesis

Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) across the cell membrane. By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone. MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%). This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen. Received: 9 December 1996 / Received revision: 31 March 1997 / Accepted: 13 April 1997     

Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Nγ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A ¹H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Characterization of mariner transposon from the genome of Himasthla elongata fluke

A novel DNA-transposon Hemar1 has been identified and characterized in the genome of the flatworm Himasthla elongata. This transposon is a representative of the mariner family, which is widely spread in eukaryotes; it belongs to the capitata subfamily and is highly homologous to the mariner element of the freshwater turbellarian worm Dugesia tigrina. The Hemar1 transposon has been established as a dispersed repeat and accounts for about 0.01% of the H. elongata genome. The identified Hemar1 element is a convenient tool for the further study of the functioning of mobile elements in the H. elongata genome.     

Complementation between mutants of Phycomyces deficient with respect to carotenogenesis

Summary White and red mutants of Phycomyces, derived from two independent wild types (yellow) by mutagenesis using nitrosoguanidine, either in a single step (26 white, 5 red mutants), or in two steps (10 white mutants, from one of the red mutants) were studied with respect to complementation in heterokaryons. The tests clearly establish the involvement of three and only three genes, here named carA, carB, and carB. The carA and the carR mutants are white, the carA mutants do not accumulate phytoene, the carB mutants do. The carR mutants are red and accumulate lycopene. The two step mutants are either carA and carR, or carB and carR double mutants. A few of the white mutants obtained in a single mutagenization step are affected in carA and carR. They may be polar mutants in an operon or accidental double mutants.     

Genetic differentiation of metallicolous and non-metallicolous Armeria maritima (Mill.) Willd. taxa (Plumbaginaceae) in Central Europe

This study investigates the genetic differentiation within the Central European Armeria maritima (Mill.) Willd. complex with special reference to the metallicolous populations using AFLP markers. Our sampling comprised all metallicolous (ssp. halleri, hornburgensis, bottendorfensis, eifeliaca, calaminaria), and non-metallicolous taxa (ssp. maritima, elongata, alpina). Geographical and genetic distances between populations were moderately positively correlated. Genetic variability of metallicolous and non-metallicolous populations was not significantly different. Lowland populations were clearly differentiated from the alpine populations. Within the lowland group metallicolous and non-metallicolous populations were not genetically differentiated. All lowland populations show a regional differentiation and close relationships to ssp. elongata. Thus, the metallicolous taxa should not be maintained as subspecies. Likewise, their treatment as varieties of a ssp. halleri s.l. is critical because this taxon cannot be consistently characterized throughout its geographical range and may be an artefact itself. If a taxonomical recognition should be considered necessary it is advisable to treat the microendemics as varieties of ssp. elongata.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996     

Taxonomic notes on Pogonieae ( Orchidaceae ): Cleistesiopsis, a new genus segregated from Cleistes , and description of two new South American species, Cleistes batistana and C. elongata

Summary  A new genus, Cleistesiopsis, was segregated from Cleistes based on morphological and molecular characteristics, and two new species of Cleistes: C. batistana and C. elongata, both occurring in the Brazilian Central Plateau (Central-Western Brazil), are described and illustrated. Furthermore, a key to genera currently recognised within Pogonieae is presented.

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Resumo  Um novo gênero, Cleistesiopsis, é segregado de Cleistes com base em caracteres morfológicos e moleculares, e duas novas espécies de Cleistes: C. batistana e C. elongata, que ocorrem no Planalto Central, no Centro-Oeste do Brasil, s?o descritas e ilustradas. Além disso, uma chave para os gêneros que atualmente comp?em a tribo Pogonieae é apresentada.