Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp.

The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.     

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

Background  

Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.     

Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.     

Screening of Malaysian indigenous microalgae for antioxidant properties and nutritional value

Fourteen indigenous microalgal samples from Malaysia were isolated, purified and cultured from fresh, brackish and marine waters. The ability of the microalgae to be natural sources of antioxidants was studied by a screening test using three antioxidant chemical assays [ferric thiocyanate (FTC), thiobarbituric acid (TBA) and 1, 1’-diphenyl-2-picrylhydrazyl (DPPH)]. The results showed that six microalgal methanolic crude extracts (Isochrysis galbana, Chaetoceros calcitrans, Scenedesmus quadricauda, Chlorella vulgaris, Nannochloropsis oculata and Tetraselmis tetrathele) were active in inhibiting the lipid peroxidation of linoleic acid. Among all the microalgae, I. galbana and C. calcitrans showed the highest antioxidant activity (>90%) in FTC and TBA assays, indicating that these microalgae might contain active compounds for protection from lipid peroxidation. Nutritional analyses were performed on microalgae with high antioxidant activities (I. galbana and C. calcitrans) in order to investigate their nutritive value. Both microalgae were found to be rich in nutrients. For examples, I. galbana had average percentage composition of protein, carbohydrate, and lipid, as 47.9 ± 2.5; 26.8 ± 0.2; 14.5 ± 1.4%, respectively, while the corresponding values for C. calcitrans were 36.4 ± 1.7; 27.4 ± 3.0; 15.5 ± 0.9%. In addition, they contained high levels of omega-3 polyunsatrated fatty acids (PUFA) (28.0% ± 0.7 in I. galbana and 28.5% ± 1.4 in C. calcitrans), omega-6 PUFA (6.5% ± 1.8 in I. galbana and 23.0% ± 2.5 in C. calcitrans) and a high composition of essential amino acids. This study illustrates that some microalgae such as I. galbana and C. calcitrans have the potential to be used as natural sources of antioxidants with high nutritional value. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Recombination,cryptic clades and neutral molecular divergence of the microcystin synthetase (<Emphasis Type="Italic">mcy</Emphasis>) genes of toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Background  

The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups.     

Evidence for Recombination in the Microcystin Synthetase (<Emphasis Type="Italic">mcy</Emphasis>) Genes ofToxic Cyanobacteria <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Bold">spp</Emphasis>

Recombination has been suggested to be an important factor for the genetic variation of bacterial genes, but few studies have dealt with intragenic recombination between the same or closely related species of cyanobacteria. Here we provide strong evidence for recombination in the microcystin synthetase (mcy) gene cluster of the toxic cyanobacteria Microcystis spp. This gene cluster contains 10 genes (mcyA to J) that encode a mixed polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) complex. mcy gene sequences were determined for four selected regions (within mcyA, D, G, and J) within the mcy gene cluster from 1 Canadian and 10 Asian toxic Microcystis and compared with previously published mcy sequences. Split decomposition analysis indicated a reticulate phylogeny of mcyA, and several potential recombination tracts of mcyA were identified by the RDP analysis and a runs test implemented in GENECONV. In contrast, no recombination was detected in the mcyD, G, and J sequences. However, discrepancies among the four mcy gene genealogies were evident from the results of independent split decomposition analyses, which were further supported by incongruence length difference (ILD) tests. Taken together, these findings suggest that both intragenic and intergenic recombination within the mcy gene cluster contributes to the genetic diversity of the mcy genes of Microcystis spp.This article contains online supplementary material.     

Characterization of the Nodularin Synthetase Gene Cluster and Proposed Theory of the Evolution of Cyanobacterial Hepatotoxins

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.     

Edible oil degradation by using yeast coculture of <Emphasis Type="Italic">Rhodotorula pacifica</Emphasis> ST3411 and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> ST3412

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.     

Spatial and temporal diversity of microcystins and microcystin-producing genotypes in Oneida Lake, NY

Oneida Lake is a shallow, eutrophic lake with a well-established cyanobacterial population with reported toxic blooms containing hepatotoxic microcystins (MC). Peak bloom events from the summers of 2002 and 2003 were analyzed to determine the principal cyanobacterial genera containing microcystin synthetase (mcy) genes. Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena and Planktothrix sp. indicated that Microcystis sp. was the dominant mcy genotype. This Microcystis clade was split into two distinct sub-clades. Bloom events contained members of both sub-clades with the higher MC concentrations found when both sub-clades were present in near equal proportions. The proportion of Microcystis containing the mcyD gene ranged from 0 to 37% of the total Microcystis population as determined by quantitative PCR (qPCR). The total concentration of Microcystis containing mcyD genes was linearly related to the concentration of MCs (r² = 0.63). The relationship between mcy genotype and physiochemical variables was examined to determine the factor(s) controlling the periodicity in MC production in Oneida Lake. Multivariate statistical analyses, used to correlate the continuous-response variables, revealed a strong relationship between chlorophyll a, MCs and total Microcystis.     

The impact of maternal experience on post-weaning survival in an endangered arctic fox population

Behavioural differences in parental care can influence offspring survival through variation in e.g. antipredator behaviour and ability to provide food. In a broad range of species, offspring survival has been found to be higher for experienced females compared to inexperienced first-time breeders. The increase in offspring survival for experienced females has mainly been explained by improved experience in providing food. In this paper, we have studied post-weaning juvenile survival in relation to maternal experience in an endangered population of arctic foxes (Vulpes lagopus) in Fennoscandia. For cubs raised by inexperienced and experienced females, the survival rate was 0.42 (CI 95% ± 0.31) and 0.87 (CI 95% ± 0.08), respectively. There was no difference in body condition between the cubs and no observations of starvation. We suggest that the difference in survival was due to lack of experience to one of the most common predators, the golden eagle (Aquila chrysaetos). Golden eagles were mainly observed on dens with litters where the females were inexperienced first-time breeders. From a conservation perspective, it is therefore important to increase adult survival through actions to enlarge the proportion of experienced breeders.     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

Integrating phylogeny,geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix

Toxic freshwater cyanobacteria form harmful algal blooms that can cause acute toxicity to humans and livestock. Globally distributed, bloom-forming cyanobacteria Planktothrix either retain or lose the mcy gene cluster (encoding the synthesis of the secondary metabolite hepatotoxin microcystin or MC), resulting in a variable spatial/temporal distribution of (non)toxic genotypes. Despite their importance to human well-being, such genotype diversity is not being mapped at scales relevant to nature. We aimed to reveal the factors influencing the dispersal of those genotypes by analyzing 138 strains (from Europe, Russia, North America and East Africa) for their (i) mcy gene cluster composition, (ii) phylogeny and adaptation to their habitat and (iii) ribosomally and nonribosomally synthesized oligopeptide products. Although all the strains from different species contained at least remnants of the mcy gene cluster, various phylogenetic lineages evolved and adapted to rather specific ecological niches (for example, through pigmentation and gas vesicle protein size). No evidence for an increased abundance of specific peptides in the absence of MC was found. MC and peptide distribution rather depended on phylogeny, ecophysiological adaptation and geographic distance. Together, these findings provide evidence that MC and peptide production are primarily related to speciation processes, while within a phylogenetic lineage the probability that strains differ in peptide composition increases with geographic distance.     

Estimating toxic cyanobacteria in a Brazilian reservoir by quantitative real-time PCR, based on the microcystin synthetase D gene

The production of microcystin toxins by cyanobacteria is an intrapopulation feature and the toxic and nontoxic genotypes can be separated only through molecular analyses targeting the mcy markers. Quantitative real-time PCR (qPCR) is a procedure that has been established, not only to detect but to specifically quantify these genotypes. In the present work, primers were designed for the mcyD region to estimate the number of cyanobacteria that are potential microcystin producers. Laboratory tests to verify the efficiency and the specificity of the primers were performed. The methodology was first established for single strain cultures and thereafter was applied in environmental water samples, from a reservoir located in the Brazilian savannah (“cerrado”). The results were very satisfactory, demonstrating the high efficiency and the specificity of the primers used, and their ability to detect different cyanobacteria genera. Of particular interest were the results showing a high proportion of toxic strains (as high as 100 %) in the environmental samples, as previously reported in another tropical system. Furthermore, the occurrence of a smaller fraction of toxic strains at high cyanobacteria densities, and of more toxic populations when fewer cyanobacteria were present, deserves further investigation. Although records of cyanobacteria blooms are very common in the tropics and suggest an increasing incidence of toxic populations, the present research is one of the few applying qPCR in a tropical environment. The results obtained here, by a technique that allows a more precise quantification and in situ follow-up of changes in toxicity, will make possible new observations of seasonal and spatial dynamics in these environments.     

Nontoxic strains of cyanobacteria are the result of major gene deletion events induced by a transposable element

Blooms that are formed by cyanobacteria consist of toxic and nontoxic strains. The mechanisms that result in the occurrence of nontoxic strains are enigmatic. All the nontoxic strains of the filamentous cyanobacterium Planktothrix that were isolated from 9 European countries were found to have lost 90% of a large microcystin synthetase (mcy) gene cluster that encoded the synthesis of the toxic peptide microcystin (MC). Those strains still contain the flanking regions of the mcy gene cluster along with remnants of the transposable elements that are found in between. The majority of the strains still contain a gene coding for a distinct thioesterase type II (mcyT), which is putatively involved in MC synthesis. The insertional inactivation of mcyT in an MC-producing strain resulted in the reduction of MC synthesis by 94 +/- 2% (1 standard deviation). Nontoxic strains that occur in shallow lakes throughout Europe form a monophyletic lineage. A second lineage consists of strains that contain the mcy gene cluster but differ in their photosynthetic pigment composition, which is due to the occurrence of strains that contain phycocyanin or large amounts of phycoerythrin in addition to phycocyanin. Strains containing phycoerythrin typically occur in deep-stratified lakes. The rare occurrence of gene cluster deletion, paired with the evolutionary diversification of the lineages of strains that lost or still contain the mcy gene cluster, needs to be invoked in order to explain the absence or dominance of toxic cyanobacteria in various habitats.     

Mitochondrial DNA reveals low population differentiation in elongate loach, <Emphasis Type="Italic">Leptobotia elongata</Emphasis> (Bleeker): implications for conservation

Elongate loach (Leptobotia elongata (Bleeker)), an endemic fish species to China, is a famous ornamental freshwater fish. Here, a comparative study of mtDNA control region (D-loop) (835 bp) sequences was performed to analyze its wild population structure and evaluate the genetic diversity for 110 individuals from five locations in the upper reaches of the Yangtze River, China. A total of 49 polymorphic sites and 45 haplotypes yielded high haplotype diversity (h = 0.952), but low nucleotide diversity (π = 0.00454) as that of many fish species. Sequence divergences between haplotypes ranged from 0.0033 ± 0.0011 to 0.0050 ± 0.0012 in intra-groups, and from 0.0037 ± 0.0.0011 to 0.0050 ± 0.0012 between groups. Significant values of Tajima’s D (−1.86383, P < 0.01) and Fu’s F _S (−25.93, P < 0.01), together with uni-modal mismatch distribution, indicated a recent genetic bottleneck or population expansion of the species. Analysis of molecular variance (AMOVA) indicated a small amount of differentiation among groups (1.7%); most of the total variation occurred within groups (98.3%). Also, there was no significant population structure (F _ST = 0.017, P > 0.05), and estimates of gene flows among groups were extremely high (Nm = 28.88), suggesting low genetic divergence between populations in the species. The lack of genetic differentiation among groups is most likely due to the combined gene flow from the downstream movement of eggs and larvae with currents and the upstream or downstream migration of adults throughout the distribution. These groups of L. elongata distributed in upper reaches of the Yangtze River should be considered as a single management unit.     

Effects of Nanoparticles on the Adhesion and Cell Viability on Astrocytes

In recent years, both pharmaceutical companies and manufacturing industries have expressed heightened interest in the potential applications of magnetic nanoparticles for therapeutic and technological purposes. Specifically, pharmaceutical companies seek to employ magnetic nanoparticles as carriers to facilitate effective drug delivery, especially in areas of the brain. Manufacturing industries desire to use these nanoparticles as ferrofluids and in magnetic resonance imaging. However, data concerning the effects of magnetic nanoparticles on the nervous system is limited. This study tested the hypotheses that nanoparticles can (1) inhibit adherence of astrocytes to culture plates and (2) cause cytotoxicity or termination of growth, both end points representing surrogate markers of neurotoxicity. Using light microscopy, changes in plating patterns were determined by visual assessment. Cell counting 4 days after plating revealed a significant decrease in the number of viable astrocytes in nanoparticle treated groups (p < 0.0001). To determine the cytotoxic effects of nanoparticles, astrocytes were allowed to adhere to culture plates and grow to maturity for 3 weeks before treatment. Membrane integrity and mitochondrial function were measured using colorimetric analysis lactate dehydrogenase (LDH) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTS), respectively. Treatment with nanoparticles did not significantly alter astrocytic LDH release (p > 0.05) in the control group (100% ± 1.56) vs the group receiving treatment (97.18% ± 2.03). However, a significant increase in MTS activity (p < 0.05) between the control (100% ± 3.65) and treated groups (112.8% ± 3.23) was observed, suggesting astrocytic mitochondrial uncoupling by nanoparticles. These data suggest that nanoparticles impede the attachment of astrocytes to the substratum. However, once astrocytes attach to the substratum and grow to confluence, nanoparticles may cause mitochondrial stress.     

Three genetically divergent lineages of the Oryx in eastern Africa: Evidence for an ancient introgressive hybridization

Phylogeographic and population genetic studies using sequence information are frequently used to infer species boundaries and history; and to assess hybridization and population level processes. In this study, partial mitochondrial DNA (mtDNA) control region (423 bp) and cytochrome b sequences (666 bp) of Oryx beisa sampled from five isolated localities in its entire current range in Africa were analyzed to investigate the extent of genetic variation and differentiation between populations. We observed high nucleotide diversity at the control region in the total sample (6.3%) but within populations, it varied considerably ranging from 1.6% to 8.1%. Population pairwise genetic differentiation was generally significantly high (ranging from F _ST = 0.15, P<0.01 to F _ST = 0.54, P<0.001). In the total sample, 29 and 12 haplotypes were observed in the control region and the cytochrome b data sets respectively. For both data sets, the haplotypes cluster into three distinct clades (sequence divergence ranged from 6.0%–12.9% to 0.8%–1.0% for the control region and cytochrome b sequences, respectively) that do not correspond to sampling locations. Two of these clades are found in the same localities (Samburu and Marsabit), which represent the O.beisa beisa subspecies, whereas the last clade represents the fringe-eared oryx (O. beisa callotis). We interpret these findings in terms of an ancient hybridization and introgression between two formerly isolated taxa of Oryx beisa.     

Initial Characterization of Micafungin Pulmonary Delivery via Two Different Nebulizers and Multivariate Data Analysis of Aerosol Mass Distribution Profiles

Pharmaceutical aerosols have been targeted to the lungs for the treatment of asthma and pulmonary infectious diseases successfully. Micafungin (Astellas Pharma US, Deerfield, IL, USA) has been shown to be an effective antifungal agent when administrated intravenously. Pulmonary delivery of micafungin has not previously been reported. In the present pilot study, we characterize the performance of two nebulizers and their potential for delivering micafungin to the lungs as well as the use of multivariate data analysis for mass distribution profile comparison. The concentration of micafungin sodium increased by 21% when delivered by the Acorn II nebulizer and by 20% when delivered by the LC Plus nebulizer, respectively, from the first to the second sampling period. The Acorn II nebulizer delivered a fine particle fraction FPF_5.8 (%<5.8 μm) of 92.5 ± 0.8 and FPF_3.3 (%<3.3 μm) of 82.3 ± 2.1 during the first sampling period. For the LC Plus nebulizer, FPF_5.8 was 92.3 ± 0.1 and FPF_3.3 was 67.0 ± 0.7 during the first sampling period. The mass median aerodynamic diameter (MMAD) increased from 1.67 ± 0.05 to 1.77 ± 0.04 μm (Acorn II nebulizer) and from 2.09 ± 0.01 to 2.20 ± 0.01 μm (Pari LC Plus nebulizer) from the first to the second sampling periods. These changes in MMAD were statistically significant by paired t test. Multivariate data analysis showed that this could be explained systematically by greater drug deposition on stages with larger cutoff sizes and reduced drug deposition on stages with smaller cutoff sizes rather than multimodal deposition or other anomalies in size distribution.     

An initial comparison of tubular netting versus tie–tie methods of cultivation for Kappaphycus alvarezii (Rhodophyta, Solieriaceae) on the south coast of Rio de Janeiro State, Brazil

A new cultivation technique for Kappaphycus alvarezii was used in the Brazilian southeastern coast (23°02′25″ S and 43°53′39″ W), the tubular netting on floating rafts. The tubular net technique (TN) was compared with the current method of tie–tie (TT). After 47 days, the daily growth rate (mean ± SD of TN and TT = 3.4 ± 0.7% day⁻¹), carrageenan yield (mean ± SD of TN and TT = 36 ± 1.3%), gel strength (mean ± SD of TN and TT = 730 ± 114.5 g cm2), and viscosity (mean ± SD of TN and TT = 350 ± 60.4 cP) did not differ between the two techniques (p > 0.05). The amount of time used to plant (TT = 30.2 ± 2.3 min and TN = 13.3 ± 3.4 min) and to harvest the seedlings (TT = 36.2 ± 2.7 min and TN = 17.8 ± 3.7 min) were lower in TN (p < 0.001). It is concluded that TN was more effective than TT, the cultivation management (time rates used to plant and harvest the seedlings) was 53.6% faster, no seedlings were lost, the cost was lower, and an estimated return in 1 year of nearly 20% more than that of the TT technique.     

Asymmetric crossing over in the spontaneous formation of large deletions in the tonB-trp region of the Escherichia coli K-12 chromosome.

The chromosomal tonB gene of Escherichia coli was used as a target for the detection of spontaneous deletion mutations. The deletions were isolated in both recA ⁺ and recA ⁻ cells, and mutants carrying large deletions were identified because they also lacked part or all of the trp operon. The frequencies of tonB-trp deletion were 1.79 × 10⁻⁹ and 1.09 × 10⁻⁹ for recA ⁺ and recA ⁻ cells, respectively. We analyzed 12 deletions from recA ⁺ and 10 from recA ⁻ cells by cloning and direct sequencing. The deletions ranged in size from 5612 bp to 15142 bp for recA ⁺ and from 5428 bp to 13289 for recA ⁻ cells. Three deletions from recA ⁺ cells and five deletions from recA ⁻ cells were found to have occurred between short sequence repeats at the termini of the deletion, leaving one copy of the repeat in the mutant sequence. Seven deletions from recA ⁺ cells and three deletions from recA ⁻ cells did not have repeats at their termini; in these cases, the DNA sequences that are adjacent to the deletion termini in the wild-type are characterized by short (2–4 bp) repeats. From these results, a model is presented for the generation of deletion mutations which involves formation of an asymmetric crossover mediated by repeated sequences of 2- to 4-bp. Received: 14 September 1998 / Accepted: 22 December 1998