Phototropins do not alter accumulation of evening-phased circadian transcripts under blue light.

The circadian system induces rhythmic variation in a suite of biochemical and physiological processes that serves to optimise plant growth in diel cycles. To be of greatest utility, these rhythmic behaviours are coordinated with regular environmental changes such as the rising and setting of the sun. Photoreceptors, and metabolites produced during photosynthesis, act to synchronise the internal timing mechanism with lighting cues. We have recently shown that phototropins help maintain robust rhythms of photosynthetic operating efficiency (?PSII or Fq'/Fm') under blue light, although rhythmic accumulation of morning-phased circadian transcripts in the nucleus was unaffected. Here we report that evening-phased nuclear clock transcripts were also unaffected. We also observe that rhythms of nuclear clock transcript accumulation are maintained in phototropin mutant plants under a fluctuating lighting regime that induced a loss of Fq'/Fm' rhythms

3'-Phosphoadenosine 5'-Phosphate Accumulation Delays the Circadian System.

The circadian system optimizes cellular responses to stress, but the signaling pathways that convey the metabolic consequences of stress into this molecular timekeeping mechanism remain unclear. Redox regulation of the SAL1 phosphatase during abiotic stress initiates a signaling pathway from chloroplast to nucleus by regulating the accumulation of a metabolite, 3'-phosphoadenosine 5'-phosphate (PAP). Consequently, PAP accumulates in response to redox stress and inhibits the activity of exoribonucleases (XRNs) in the nucleus and cytosol. We demonstrated that osmotic stress induces a lengthening of circadian period and that genetically inducing the SAL1-PAP-XRN pathway in plants lacking either SAL1 or XRNs similarly delays the circadian system. Exogenous application of PAP was also sufficient to extend circadian period. Thus, SAL1-PAP-XRN signaling likely regulates circadian rhythms in response to redox stress. Our findings exemplify how two central processes in plants, molecular timekeeping and responses to abiotic stress, can be interlinked to regulate gene expression

Pitfalls using tributyrin agar screening to detect lipolytic activity in metagenomic studies

The metagenomics approach is an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms within diverse environments. In this study, we constructed a metagenomic library using a South African deep mine biofilm sample. The library was screened forlipolytic activity using LB Tributyrin (TLB). Although we were able to identify 3 diverse esterase enzymes, we found that 70% of the obtained sequence data revealed the presence of enzymes and genes completely unrelated to that of lipolytic enzymes thereby highlighting the limitation of screening with TLB

Analysing the role of SAL1/PAP retrograde signalling within the circadian system of Arabidopsis thaliana

Plants have developed an internal timing mechanism, the circadian system, that serves to synchronise physiological and metabolic functions with daily, predictable cues such as dawn and dusk. This endogenous oscillator is comprised of biochemical and transcriptional rhythms that are entrained by environmental signals, particularly light and temperature, through the action of input pathways. The circadian system provides plants with an adaptive advantage, and techniques that allow in vivo monitoring of circadian rhythms give valuable insights into the components and mechanisms employed by plants to optimally respond to abiotic signals. This study shows that chlorophyll a fluorescence imaging can be used to describe circadian rhythms of PSII operating efficiency (Fq’/Fm’) in the chloroplasts of Arabidopsis thaliana. These circadian rhythms in Fq’/Fm’ are influenced by the well-defined rhythmic transcriptional feedback loops that comprise the central oscillator in the nucleus, and are maintained under constant blue light by the action of phototropin photoreceptors. Using chlorophyll a fluorescence imaging, the chloroplast-localised enzyme SAL1 was identified as impacting circadian oscillations both in chloroplasts and in the nucleus. SAL1 is a redox-sensitive component of the SAL1-PAP-XRN retrograde signalling pathway, and influences nuclear gene expression in response to stress by modulating the levels of its substrate, 3’-phosphoadenosine 5’-phosphate (PAP). PAP accumulates in chloroplasts under abiotic stress and inhibits the activity of 5’→3’ exoribonucleases (XRNs). This study shows that genetically inducing the SAL1-PAP-XRN pathway in plants lacking SAL1 function induces a long circadian period in a blue light-dependent manner. Application of exogenous PAP or osmotic stress lengthens circadian period, and period lengthening correlates with increases in endogenous PAP levels. Furthermore, plants lacking functional XRNs exhibit a similar long circadian period phenotype. The SAL1-PAP-XRN pathway is therefore proposed to regulate nuclear circadian rhythms in response to changes in chloroplast redox poise, and serves as a possible link between molecular timekeeping and abiotic stress response mechanisms

Focusing on the nuclear and subnuclear dynamics of light and circadian signalling

Circadian clocks provide organisms the ability to synchronise theirinternal physiological responses with the external environment. Thisprocess, termed entrainment, occurs through the perception of internaland external stimuli. As with other organisms, in plants the perception oflight is a critical for the entrainment and sustainment of circadianrhythms. Red, blue, far-red and UV-B light is perceived by the oscillatorthrough the activity of photoreceptors. Four classes of photoreceptorssignal to the oscillator: phytochromes, cryptochromes, UVR8 and LOV-KELCH domain proteins. In most cases, these photoreceptors localise tothe nucleus in response to light and can associate to subnuclearstructures to initiate downstream signalling. In this review, we willhighlight the recent advances made in understanding the mechanismsfacilitating the nuclear and subnuclear localisation of photoreceptors andthe role these subnuclear bodies have in photoreceptor signalling,including to the oscillator. We will also highlight recent progress that hasbeen made in understanding the regulation of the nuclear andsubnuclear localisation of components of the plant circadian clock

A deep gold mine metagenome as a source of novel esterases

New sources of enzymes for biotechnological applications are continually being sought for. While diverse microbial ecosysyems have been demonstrated in the deep subsurfaces, deep mines provide easy access to these specialist communities. Therefore, the aim of this study was to assess a deep mine biofilm as a source of novel esterase enzymes. Biofilm was collected from the Beatrix Mine in South Africa, at a depth of 808 m. Assessment of the diversity revealed a group of previously uncultured bacteria and archaea. A metagenome library was screened for esterolytic activity, producing two esterolytic clones: a phospholipase patatin protein and an isochorismatase family protein. The isochorismatase family protein contained the catalytic Asp and Cys but not the Arg, which is considered as important for catalysis. The patatin showed 55% similarity to its closest relative; the patatin family protein from Plesiocystis pacifica. The expressed patatin displayed a preference for the C6 ester and was maximally active at pH 8 and 30°C. This study reported that screening of a relatively small metagenome library from the deep mine biofilm provided two esterolytic clones, which differed from their known counterparts. This indicates that the deep mine ecosystems contain an untapped resource of novel and potentially useful enzymes which may have applications in chemical syntheses.Key words: Metagenome library, functional screening, lipolytic activity, patatin, isochorismatas

A deep gold mine metagenome as a source of novel esterases.

New sources of enzymes for biotechnological applications are continually being sought for. While diverse microbial ecosysyems have been demonstrated in the deep subsurfaces, deep mines provide easy access to these specialist communities. Therefore, the aim of this study was to assess a deep mine biofilm as a source of novel esterase enzymes. Biofilm was collected from the Beatrix Mine in South Africa, at a depth of 808 m. Assessment of the diversity revealed a group of previously uncultured bacteria and archaea. A metagenome library was screened for esterolytic activity, producing two esterolytic clones: a phospholipase patatin protein and an isochorismatase family protein. The isochorismatase family protein contained the catalytic Asp and Cys but not the Arg, which is considered as important for catalysis. The patatin showed 55% similarity to its closest relative; the patatin family protein from Plesiocystis pacifica. The expressed patatin displayed a preference for the C6 ester and was maximally active at pH 8 and 30°C. This study reported that screening of a relatively small metagenome library from the deep mine biofilm provided two esterolytic clones, which differed from their known counterparts. This indicates that the deep mine ecosystems contain an untapped resource of novel and potentially useful enzymes which may have applications in chemical syntheses

SAL1-PAP retrograde signalling extends circadian period by reproducing the loss of exoribonuclease (XRN) activity

Plants have developed an internal timing mechanism, the circadian system, that serves to synchronise physiological and metabolic functions with daily cues such as dawn and dusk, and provides plants with an advantage in adapting to changing and challenging conditions. We have recently shown that the SAL1-PAP-XRN retrograde signalling pathway, which is proposed to regulate plant responses under stress conditions, also acts within the circadian system. Here we provide further evidence of circadian regulation by SAL1-PAP-XRN signalling, thereby affirming a link between molecular timekeeping and abiotic stress response mechanisms

Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species

BACKGROUND: Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms. RESULTS: Using Meiothermus silvanus DSM 9946 as a reference genome, high level of coordinated rearrangements has been observed in extremely thermophilic Thermus that may imply existence of yet unknown evolutionary forces controlling adaptive re-organization of whole genomes of thermo-extremophiles. However, no remarkable differences were observed across species on distribution of functionally related genes on the chromosome suggesting constraints imposed by metabolic networks. The metabolic network exhibit evolutionary pressures similar to levels of rearrangements as measured by the cross-clustering index. Using stratigraphic analysis of donor-recipient, intensive gene exchanges were observed from Meiothermus species and some unknown sources to Thermus species confirming a well established DNA uptake mechanism as previously proposed. CONCLUSION: Global genome rearrangements were found to play an important role in the evolution of Thermus bacteria at both genomic and metabolic network levels. Relatively higher level of rearrangements was observed in extremely thermophilic Thermus strains in comparison to the thermo-tolerant Thermus scotoductus. Rearrangements did not significantly disrupt operons and functionally related genes. Thermus species appeared to have a developed capability for acquiring DNA through horizontal gene transfer as shown by the donor-recipient stratigraphic analysis.http://www.biomedcentral.com/bmcgenomics/am201

Bacterial diversity of biofilm samples from deep mines in South Africa

The Au, Pt and diamond mines of South Africa provide access to microorganism bearing fluids emanating from fractures at depths ranging from 0.7 to 3.2 km. Due to the unique characteristic of mine environment as demonstrated by extreme pH, pressure, temperatureand/or salinity, it is anticipated that it could hold the promise for novel gene sequences and hence gene products of industrial and pharmaceutical importance. To provide insight into themicrobial diversity of mines in South Africa, biofilm samples were collected from Goldfield and diamond mines and their bacterial diversity determined using molecular approaches. 16S rRNA genes were amplified from DNA extracted from these samples using polymerase chain reaction with universal bacterial primers 27F (5’- AGA GTT TGA TCM TGG CTC AG-3’) and 1492R (5’- GGT TAC CTT GTT ACG ACT T-3’). Metagenomic clone libraries were constructed and restriction fragment length polymorphism (RFLP) analysis of >100 derivedclones resulted in four major restriction patterns from which 40 clones were chosen for sequencing. More than half (53%) of the sequences were affiliated with the bacterial phylum Proteobacteria, forty-one percent (41%) of the sequences with yet uncultured bacteria andthe phyla Firmicutes and Planctomycetes were accounted for by 4% and 2% of the sequences respectively. DGGE analysis of PCR-amplified 16S rRNA genes showed characteristic fingerprints for each sample. The differences in community structure observed account for the uniqueness of each of the mines with respect to its microbial diversity