Nitrogen and phosphorus in soil solutions and drainage streams in Upper Teesdale, northern England: implications of organic compounds for biological nutrient limitation.

The availability of nitrogen (N) and phosphorus (P) to organisms is a fundamental control on the ecology of uplands in the British Isles, yet there has been relatively little consideration of the role of organic forms of these nutrients. N and P fractions in soil solutions and drainage streams were determined at monthly intervals over 1 year from Widdybank Fell, Upper Teesdale National Nature Reserve, an upland area of northern England. Nitrogen was present in inorganic and organic forms at relatively high concentrations, whereas P was mainly organic, with inorganic P concentrations near the detection limit (1 microg Pl(-1)). The highest concentrations of organic P occurred in 'pulses' during the spring. These pulses may have originated in the soil following drying and rewetting cycles, and appear to demonstrate hydrochemical connectivity between soils and streams. Seasonal changes in N:P ratio were estimated in various ways to assess when the availability of these elements was likely to influence organisms. Based on inorganic nutrients alone, marked P limitation would be expected throughout the year, but inclusion of organic nutrients suggests that some communities may become N-limited during the spring. This agrees with known seasonal changes in the biology of the cyanobacterium Rivularia, which dominates the main drainage streams. Our data highlight the importance of including organic nutrients when assessing biological nutrient limitation.

Conclusions to the special issue of Science of the Total Environment concerning 'The water quality of UK rivers entering the North Sea'.

This paper presents an overview of the water quality functioning of eastern UK rivers draining into the North Sea. It summarises the results of three special issues of Science of the Total Environment and related work and complements a companion bibliography (Neal and Turner, 2000, this volume). The rivers are described in straightforward broad terms, and the reader is guided via the bibliography to the special volumes for the detailed findings. The water quality is shown to be highly variable over space and time. The changes over the past 300 years reflect first the transition from a rural society to the major impact of the industrial revolution and subsequently to one of the worlds' most successful environmental cleanups. The present water quality reflects the relative impacts of diffuse agricultural sources, urban and industrial point source discharges together with background inputs associated with geology: all these can be modified by within-river processes associated with physical mixing, biology and inorganic reactions. The work is set within the context of water quality, modelling and flux outputs to the North Sea and contemporary and future environmental research and management needs. A range of environmental impact studies is included. The detailed information is now available on a major and easily accessible database.

Arthrospira ('Spirulina') strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer.

We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira ('Spirulina') cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 51 tested cultures. The strain Spirulina laxissima SAG 256.80 was included as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by amplified ribosomal DNA restriction analysis, and thus the internally transcribed spacer was selected as molecular taxonomic marker. The internally transcribed spacer sequences situated between the 16S and the 23S rRNA were amplified by polymerase chain reaction and yielded amplicons of about 540 bp. Direct use of cells for polymerase chain reaction seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of bovine serum albumin in the polymerase chain reaction mix. The amplicons were digested with four restriction enzymes (EcoRV, Hhal, Hinfl, Msel) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology.

Biology of the Humber rivers.

An overview of the literature is presented on the biology of the rivers entering the Humber, eastern England, together with some of their tributaries. Particular emphasis is given to dynamic aspects, including transport and movement within rivers, movement between rivers, processes within rivers and long-term changes.

Singular over-representation of an octameric palindrome, HIP1, in DNA from many cyanobacteria.

An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome). The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries. DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1. Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage. To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced. HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides. An oligonucleotide including HIP1 was also tested in PCR. Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns. However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.

Construction of Zn2+/Cd2+ hypersensitive cyanobacterial mutants lacking a functional metallothionein locus.

Eukaryotic metallothioneins (MTs) have been extensively studied, but the precise functions of most of these molecules are not yet fully understood. Prokaryotes are often more tractable for the analysis of gene function and we report here the generation of mutants of Synechococcus PCC 7942 (strain R2-PIM8) deficient in the MT locus, smt. Viability of these mutants, designated R2-PIM8 (smt), reveals that prokaryotic MT performs no "vital" role (such as donation of metals to metallo-proteins) in Synechococcus. R2-PIM8 (smt) has reduced (approximately 5-fold) tolerance to elevated Zn2+, with detectable hypersensitivity to Cd2+. Restoration of Zn2+ tolerance was used as a selectable marker to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment containing an uninterrupted smt locus. These smt-complemented cells also exhibited restored Cd2+ tolerance. Hypersensitivity to Cu2+ was not detected in R2-PIM8(smt) indicating independence of Cu2+ resistance from smt mediated metal (Zn2+/Cd2+) tolerance.

Deletion within the metallothionein locus of cadmium-tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1).

Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal-tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance. Following the characterization of this locus, the altered smt region has now been isolated from a Cd-tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion within smtB, which encodes a trans-acting repressor of smt transcription. Two identical palindromic octanucleotides (5'-GCGATC-GC-3') traverse both borders of the excised element. This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL/DDBJ DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c. 664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed.

Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium.

Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described. To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture. An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd). Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd). Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines. Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived. As a control, another gene, psaE, was also monitored in these cell lines. There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines.

Cell-bound and extracellular phosphatase activities of cyanobacterial isolates.

Fifty cyanobacterial strains (10 genera) were tested in batch culture for their ability to use organic phosphorus compounds (1 mg liter(-1) P) as their sole P source. Two monoesters, Na2-ß-glycerophosphate and π-nitrophenyl phosphate (πNPP), supported growth of all strains, and the diester bis-π-nitrophenyl phosphate (bis-π-NPP) and herring sperm DNA supported almost all strains. ATP was either a very favorable or poor P source and failed to support growth of nine strains, seven of which were Rivulariaceae with trichomes ending in a hair or long tapered region. Phytic acid was in general the least favorable P source.P-limited cultures grown initially with inorganic phosphate to conditions of P limitation were also tested for cell-bound and extracellular phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities at two pH values (7.6, 10.3) using πNPP and bis-πNPP as substrates. Cell-bound PMEase was inducible in all strains and cell-bound PDEase in most strains. Most showed extracellular PMEase, but not extracellular PDEase. The highest values (µM πNPP or bis-πNPP hydrolyzed mg dry weight(-1) hour(-1)) all occurred in strains ofGloeotrichia as follows: cell-bound PMEase at pH 7.6, 2.7 µM in strain D602; cell-bound PMEase at pH 10.3, 5.2 µM in D602; extracellular PMEase at pH 7.6, 0.73 µM in D281; extracellular PMEase at pH 10.3, 6.6 µM in D281; cell-bound PDEase at 7.6, 0.40 µM in D613; cell-bound PDEase at pH 10.3, 1.0 µM in D613.The results were compared to see if they indicated possible relationships between phosphatase activity and taxonomic or ecological grouping. The following differences were significant (P<0.05). Rivulariaceae produced higher yields than filamentous non-Rivulariaceae with ß-glycerophosphate, πNPP, and DNA. Rivulariaceae with the ability to form hairs in culture showed poorer growth in ATP than non-hair-forming Rivulariaceae, but were more effective at utilizing phytic acid. Strains from calcareous environments had higher PMEase activity at pH 10.3 than strains from noncalcareous environments (P<0.01).

Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants.

Phosphomonoesterase activity of the cyanobacterium (blue-green alga) Calothrix parietina.

Cellular and extracellular phosphomonoesterase activities were compared in Calothrix parietina D550, a strain whose original environment has been studied in detail. Activity in both fractions became detectable at about the same stage in batch culture. Differences in the influence of environmental factors between the two were slight, suggesting a common origin. The optimum temperatures for cellular and extracellular activities were 40 degrees C and 30 degrees C, respectively, and the upper limits for detectable activity were 80 degrees C and 65 degrees C. The pH optimum for both cellular and extracellular activity was 10.0-10.2. When P-limited cultures were tested with p-nitrophenyl phosphate (pNPP) as substrate, Km values for cellular and extracellular activities were 43 and 33 microM pNPP, respectively. Eleven ions were tested for their influence on activity. In most cases the effect was low or negligible at concentrations likely to be present in nature or freshwater laboratory media. Where obvious effects occurred, these were usually apparent at lower concentrations with extracellular than cellular activity. One mM Ca led to a 40% increase in extracellular activity in comparison with 0.1 mM Ca, but had no effect on cellular activity. However, inorganic phosphate, which had a marked inhibitory effect at concentrations above 10 microM, brought about a similar response with cellular and extracellular activities (approximately 60% decrease with 100 microM).

Nitrogen fixation by blue-green algal communities in the intertidal zone of the lagoon of Aldabra Atoll.

All eight types of blue-green algal community sampled from the intertidal sediments of the lagoon of Aldabra showed detectable rates of acetylene reduction, and six of them showed at least some examples of rates considered by previous authors to be rapid. When the reduction rates are related to the chlorophyll a contents of the communities, the maximum rates recorded come in the following order: Hyella balani, Scytonema sp., Hyella balani-Schizothrix sp., Calothrix crustacea, Rivularia sp., Microcoleus chthonoplastes, Hyella balani-purple sulphur bacteria, Pleurocapsa-Chroococcus. With the exception of the mixed Hyella balani-purple sulphur bacterial community, the rate of acetylene reduction was in all experiments greater in the light than in the dark, the difference being significant (P<0.05) in the majority of cases. The rates were similar in all experiments whether incubation was carried out with sea or brackish water. It seems probable that nitrogen fixation by blue-green algal communities makes an important addition to the lagoon ecosystem, and that not only heterocystous, but also some non-heterocystous, species are involved. Among the latter, the data for Hyella balani and Microcoleus chthonoplastes are especially convincing.

Effect of pH on tolerance of Hormidium rivulare to zinc and copper.

The toxicity of zinc to a population of Hormidium rivulare isolated from an acid mine drainage was shown to be least at the optimum pH range for the growth of the alga, pH 3.5-4.0; toxicity increases markedly at higher pH values. Calcium clearly antagonizes the toxicity of zinc. Populations of H. rivulare isolated from higher pH values and which are resistant to zinc, are also especially resistant to low pH values, although they are unlikely ever to encounter such values naturally. Nevertheless raised levels of calcium bring about only a slight improvement of growth at very low pH values in the absence of zinc, so the mechanisms of pH and zinc tolerance are not the same. Although the acid stream population grows in the field in an environment with rather similar levels of zinc and copper, copper is less toxic than zinc at pH 3.5, but much more toxic than zinc at pH 6.0.