Enhanced glacial discharge from the eastern Antarctic Peninsula since the 1700s associated with a positive Southern Annular Mode.

The Antarctic Peninsula Ice Sheet is currently experiencing sustained and accelerating loss of ice. Determining when these changes were initiated and identifying the main drivers is hampered by the short instrumental record (1992 to present). Here we present a 6,250 year record of glacial discharge based on the oxygen isotope composition of diatoms (δ18Odiatom) from a marine core located at the north-eastern tip of the Antarctic Peninsula. We find that glacial discharge - sourced primarily from ice shelf and iceberg melting along the eastern Antarctic Peninsula - remained largely stable between ~6,250 to 1,620 cal. yr BP, with a slight increase in variability until ~720 cal. yr. BP. An increasing trend in glacial discharge occurs after 550 cal. yr BP (A.D. 1400), reaching levels unprecedented during the past 6,250 years after 244 cal. yr BP (A.D. 1706). A marked acceleration in the rate of glacial discharge is also observed in the early part of twentieth century (after A.D. 1912). Enhanced glacial discharge, particularly after the 1700s is linked to a positive Southern Annular Mode (SAM). We argue that a positive SAM drove stronger westerly winds, atmospheric warming and surface ablation on the eastern Antarctic Peninsula whilst simultaneously entraining more warm water into the Weddell Gyre, potentially increasing melting on the undersides of ice shelves. A possible implication of our data is that ice shelves in this region have been thinning for at least ~300 years, potentially predisposing them to collapse under intensified anthropogenic warming.

Organic and mineral imprints in fossil photosynthetic mats of an East Antarctic lake.

Lacustrine microbial mats in Antarctic ice-free oases are considered modern analogues of early microbial ecosystems as their primary production is generally dominated by cyanobacteria, the heterotrophic food chain typically truncated due to extreme environmental conditions, and they are geographically isolated. To better understand early fossilization and mineralization processes in this context, we studied the microstructure and chemistry of organo-mineral associations in a suite of sediments 50-4530 cal. years old from a lake in Skarvsnes, Lützow Holm Bay, East Antarctica. First, we report an exceptional preservation of fossil autotrophs and their biomolecules on millennial timescales. The pigment scytonemin is preserved inside cyanobacterial sheaths. As non-pigmented sheaths are also preserved, scytonemin likely played little role in the preservation of sheath polysaccharides, which have been cross-linked by ether bonds. Coccoids preserved thylakoids and autofluorescence of pigments such as carotenoids. This exceptional preservation of autotrophs in the fossil mats argues for limited biodegradation during and after deposition. Moreover, cell-shaped aggregates preserved sulfur-rich nanoglobules, supporting fossilization of instable intracellular byproducts of chemotrophic or phototrophic S-oxidizers. Second, we report a diversity of micro- to nanostructured CaCO3 precipitates intimately associated with extracellular polymeric substances, cyanobacteria, and/or other prokaryotes. Micro-peloids Type 1 display features that distinguish them from known carbonates crystallized in inorganic conditions: (i) Type 1A are often filled with globular nanocarbonates and/or surrounded by a fibrous fringe, (ii) Type 1B are empty and display ovoid to wrinkled fringes of nanocrystallites that can be radially oriented (fibrous or triangular) or multilayered, and (iii) all show small-size variations. Type 2 rounded carbonates 1-2 µm in diameter occurring inside autofluorescent spheres interpreted as coccoidal bacteria may represent fossils of intracellular calcification. These organo-mineral associations support organically driven nanocarbonate crystallization and stabilization, hence providing potential markers for microbial calcification in ancient rocks.

A technical platform for generating reproducible expression data from Streptomyces coelicolor batch cultivations.

Streptomyces coelicolor, the model species of the genus Streptomyces, presents a complex life cycle of successive morphological and biochemical changes involving the formation of substrate and aerial mycelium, sporulation and the production of antibiotics. The switch from primary to secondary metabolism can be triggered by nutrient starvation and is of particular interest as some of the secondary metabolites produced by related Streptomycetes are commercially relevant. To understand these events on a molecular basis, a reliable technical platform encompassing reproducible fermentation as well as generation of coherent transcriptomic data is required. Here, we investigate the technical basis of a previous study as reported by Nieselt et al. (BMC Genomics 11:10, 2010) in more detail, based on the same samples and focusing on the validation of the custom-designed microarray as well as on the reproducibility of the data generated from biological replicates. We show that the protocols developed result in highly coherent transcriptomic measurements. Furthermore, we use the data to predict chromosomal gene clusters, extending previously known clusters as well as predicting interesting new clusters with consistent functional annotations.

Cyanobacteria from benthic mats of Antarctic lakes as a source of new bioactivities.

AIMS: To exploit the cyanobacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes for the discovery of novel antibiotic and antitumour activities. METHODS AND RESULTS: In all, 51 Antarctic cyanobacteria isolated from benthic mats were cultivated in the laboratory by optimizing temperature, irradiance and mixing. Productivity was generally very low (

Biogeological Raman spectroscopic studies of Antarctic lacustrine sediments.

Analysis of lacustrine sediments is an accepted method for deciphering the palaeoenvironment of a lake's catchment area, as each strata of the sediment gives information about the rock type it was eroded from and also the state of the lake, i.e. oxic or anoxic. Antarctica has long been accepted as a putative analogue for Mars, so the analysis of Antarctic material may give results that can be compared to sediments on Mars. Raman spectroscopy has been selected as the method of analysis as it does not destroy the sample, can be used in situ and requires very little sample preparation. It is a suitable method for analysing both inorganic and organic matter and a miniature spectrometer is currently being developed for use in the field. The results from the spectrometers can serve as a guide for analysing sediments on Mars. It has been shown that Raman spectroscopy can detect and differentiate between oxic and anoxic sediments. Both 1064 and 785 nm wavelengths are suitable for laser excitation of organic and inorganic matter.

Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA.

In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.

Primary metabolism and its control in streptomycetes: a most unusual group of bacteria.

Streptomycetes are Gram-positive bacteria with a unique capacity for the production of a multitude of varied and complex secondary metabolites. They also have a complex life cycle including differentiation into at least three distinct cell types. Whilst much attention has been paid to the pathways and regulation of secondary metabolism, less has been paid to the pathways and the regulation of primary metabolism, which supplies the precursors. With the imminent completion of the total genome sequence of Streptomyces coelicolor A3(2), we need to understand the pathways of primary metabolism if we are to understand the role of newly discovered genes. This review is written as a contribution to supplying these wants. Streptomycetes inhabit soil, which, because of the high numbers of microbial competitors, is an oligotrophic environment. Soil nutrient levels reflect the fact that plant-derived material is the main nutrient input; i.e. it is carbon-rich and nitrogen- and phosphate-poor. Control of streptomycete primary metabolism reflects the nutrient availability. The variety and multiplicity of carbohydrate catabolic pathways reflects the variety and multiplicity of carbohydrates in the soil. This multiplicity of pathways has led to investment by streptomycetes in pathway-specific and global regulatory networks such as glucose repression. The mechanism of glucose repression is clearly different from that in other bacteria. Streptomycetes feed by secreting complexes of extracellular enzymes that break down plant cell walls to release nutrients. The induction of these enzyme complexes is often coordinated by inducers that bear no structural relation to the substrate or product of any particular enzyme in the complex; e.g. a product of xylan breakdown may induce cellulase production. Control of amino acid catabolism reflects the relative absence of nitrogen catabolites in soil. The cognate amino acid induces about half of the catabolic pathways and half are constitutive. There are reduced instances of global carbon and nitrogen catabolite control of amino acid catabolism, which again presumably reflects the relative rarity of the catabolites. There are few examples of feedback repression of amino acid biosynthesis. Again this is taken as a reflection of the oligotrophic nature of the streptomycete ecological niche. As amino acids are not present in the environment, streptomycetes have rarely invested in feedback repression. Exceptions to this generalization are the arginine and branched-chain amino acid pathways and some parts of the aromatic amino acid pathways which have regulatory systems similar to Escherichia coli and Bacillus subtilis and other copiotrophic bacteria.

Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes.

This is the first report of generalized transduction in the gram-positive, food-borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37 degrees C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well-characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (phiLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(phiCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.

Microwave endometrial ablation: development, clinical trials and outcomes at three years.

OBJECTIVE: To describe the introduction of microwave endometrial ablation to clinical practice, and to report the outcomes three years after endometrial ablation. DESIGN: A clinical trial using prototype microwave endometrial ablation equipment. SETTING: District general hospital. POPULATION: Forty-three women with completed families and with failed medical management for menorrhagia were treated with microwave endometrial ablation between October 1994 and April 1995. MAIN OUTCOME MEASURES: A statement of perceived menstrual loss and satisfaction supported by a menstrual symptom questionnaire score. Dysmenorrhoea was graded as a measure of described severity. Treatment time. RESULTS: Forty-three women had a total of 46 treatments. Mean treatment time: n = 43, was 141 seconds (50-310). Amenorrhoea: n = 16; 37.2%. Very light periods/discharge: n = 11; 25.6%. Improved periods and woman satisfied: n = 9; 20.9%. Improved periods and woman not satisfied: n = 1; 2.3%. Overall satisfaction at three years is 83.7%. Moderate (55.8%) or severe (27.9%) dysmenorrhoea pre-operatively had improved to 11.6% and 6.8% respectively at three years. Three re-treatments and four hysterectomies will be discussed. CONCLUSIONS: Microwave endometrial ablation is a new treatment for dysfunctional uterine bleeding using the application of microwave energy to the endometrium. This results in a rapid but restricted depth of intrauterine heating avoiding hysteroscopic fluid, operative haemorrhage and earthing risks. The technique is simple to learn and perform. Women report a high level of satisfaction three years after microwave endometrial ablation.

The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression.

Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.

Light-induced carotenogenesis in Myxococcus xanthus: light-dependent membrane sequestration of ECF sigma factor CarQ by anti-sigma factor CarR.

Light-induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon. CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase. CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark. CarS is required for the light-dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis. Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR. Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR. The severity of the constitutive phenotype depended on the distance between the uncoupled genes. When expressed in M. xanthus, a CarR:beta-galactosidase fusion protein disappeared in the light. We propose that anti-sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.

Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase.

The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.

Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region.

The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence-specific DNA-binding motifs. The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response ('heat-shock') promoters.

Light-induced carotenogenesis in Myxococcus xanthus: genetic analysis of the carR region.

Carotenogenesis is light-inducible in the non-photosynthetic, Gram-negative, bacterium Myxococcus xanthus. We report the characterization of the carR region which controls this phenomenon. Insertion of transposon Tn5 close to the carR region caused a dominant, carotenoid-constitutive mutation because of the presence of a constitutive, outward-reading promoter in the IS50L component of Tn5. In wild-type cells, a powerful, tightly-regulated, light-inducible promoter directs the transcription of two genetic functions. One of these functions is to activate transcription of the genetically unlinked carB gene, which is involved in carotenoid synthesis. The second function (carR) regulates the light-inducible promoter. We also report the mapping of two carotenoid constitutive mutations to the previously characterized carA locus.

Molecular genetic analysis of proline and tryptophan biosynthesis in Streptomyces coelicolor A3(2): interaction between primary and secondary metabolism--a review.

We studied the control of proline metabolism and tryptophan biosynthesis in Streptomyces coelicolor A3(2), because proline is involved in secondary metabolism [undecylprodigiosin (Red) biosynthesis] whilst tryptophan, to our knowledge, is not. Proline transport was constitutive in wild-type cells, as were the enzymes of proline catabolism. When we analysed proline biosynthesis, we discovered that growth in the presence of proline stimulated rather than repressed the biosynthetic genes. We isolated proline transport mutants and to our surprise discovered that such strains overproduced Red. As well as losing the ability to transport proline, they had lost, to differing extents, the ability to degrade proline. However, proline biosynthesis appeared to be unaffected. It appears that proline anabolism and catabolism in S. coelicolor A3(2) is in a state of dynamic equilibrium and that if this balance is disturbed, Red biosynthesis can act as a sink for excess proline. We cloned the trpD and the trpCBA clusters of S. coelicolor A3(2) and identified a promoter within the latter cluster. This promoter appeared not to be regulated by the presence or absence of exogenous tryptophan, but rather by the growth phase and/or the growth rate of the culture. It appears, therefore, that an amino acid biosynthetic pathway that is apparently not involved in secondary metabolism in the streptomycete is regulated at the genetic level--not by feedback repression, but rather by the overall physiological state of the cell.

Comparison of beta-galactosidase production by two inducible promoters in Myxococcus xanthus.

The inducibility of two promoter systems, one heterologous and one homologous, has been assessed in the Gram-negative bacterium Myxococcus xanthus. The heterologous system involved the hybrid tac promoter and the presence of lacIq, the lac repressor from Escherichia coli. This system is inducible in its natural host with isopropyl-beta-D-thiogalactopyranoside (IPTG). The homologous promoter system involves the light-inducible carQRS promoter, which is normally involved in the expression of the regulators of the light-inducible light-protective carotenoid synthesis regulon in M. xanthus. In each case, promoter activity and strength was assayed using the E. coli gene lacZ. In our constructs, which were present in a single copy in the M. xanthus chromosome, the carQRS promoter yielded at least a 47-fold increase in beta-galactosidase production upon light induction, whilst IPTG increased by 8-fold the amount of enzyme produced under the control of the ptac-lacIq system. Regulation by the latter was significantly higher than that obtained with the unmodified lacZ promoter.

Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters.

It was shown that the Escherichia coli lacZ gene could be expressed in the cyanobacterium Synechococcus R2 PCC7942 both as a plasmid-borne form and also integrated into the chromosome. A promoterless form of the lacZ gene was constructed and used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination. Synechococcus R2 promoter-lacZ gene fusions were then used to identify CO2-regulated promoters, by quantitatively assessing beta-galactosidase activity under high and low CO2 conditions using a fluorescence assay. Several promoters induced under low CO2 conditions were detected.

Isolation and genetic analysis of Caulobacter mutants defective in cell shape and membrane lipid synthesis.

In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.

Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.