The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

<p>Abstract</p> <p>Background</p> <p>Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC.</p> <p>Results</p> <p>The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession <ext-link ext-link-type="gen" ext-link-id="#EU386360">#EU386360</ext-link>), cloned and expressed in <it>E. coli</it>. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases.</p> <p>Conclusion</p> <p>We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.</p

Localizer:fast, accurate, open-source, and modular software package for superresolution microscopy

We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies

Priming in the Microbial Landscape: Periphytic Algal Stimulation of Litter-Associated Microbial Decomposers

Microbial communities associated with submerged detritus in aquatic ecosystems often comprise a diverse mixture of autotrophic and heterotrophic microbes, including algae, bacteria, protozoa, and fungi. Recent studies have documented increased rates of plant litter mass loss when periphytic algae are present. We conducted laboratory and field experiments to assess potential metabolic interactions between natural autotrophic and heterotrophic microbial communities inhabiting submerged decaying plant litter of Typha angustifolia and Schoenoplectus acutus. In the field, submerged plant litter was either exposed to natural sunlight or placed under experimental canopies that manipulated light availability and growth of periphytic algae. Litter was collected and returned to the laboratory, where algal photosynthesis was manipulated (light/dark incubation), while rates of bacterial and fungal growth and productivity were simultaneously quantified. Bacteria and fungi were rapidly stimulated by exposure to light, thus establishing the potential for algal priming of microbial heterotrophic decay activities. Experimental incubations of decaying litter with 14C‐ and 13C‐bicarbonate established that inorganic C fixed by algal photosynthesis was rapidly transferred to and assimilated by heterotrophic microbial decomposers. Periphytic algal stimulation of microbial heterotrophs, especially fungal decomposers, is an important and largely unrecognized interaction within the detrital microbial landscape, which may transform our current conceptual understanding of microbial secondary production and organic matter decomposition in aquatic ecosystems

Evalution of the Efficacy of the Photosystem II Inhibitor DCMU in Periphyton and Its Effects On Nontarget Microorganisms and Extracellular Enzymatic Reactions

We examined the efficacy of the photosystem II inhibitor 3-(3,4-diclorophenyl)-1,1-dimethyl urea (DCMU) for inhibition of algal photosynthesis in periphyton associated with submerged decomposing litter of Typha angustifolia. We also investigated the possible nontarget effects of DCMU exposure on heterotrophic microorganisms (i.e., bacteria and fungi) and extracellular enzyme activity associated with decaying litter. Standing-dead Typha leaf litter was submerged for 34 and 73 d, returned to the laboratory, and used for controlled laboratory experiments that examined the effect of DCMU on algal ([14C]bicarbonate, pulse-amplitude modulated fluorometry), bacterial ([3H]leucine), and fungal ([14C]acetate) production. Simultaneous assays also were conducted to examine the effect of DCMU on the activities of 4 extracellular enzymes (β-glucosidase, β-xylosidase, leucine-aminopeptidase, and phosphatase). DCMU significantly inhibited algal photosynthesis in light-exposed periphyton (p always \u3c 0.0003), with strong inhibitory effects occurring within 5 min after exposure to DCMU. In contrast, DCMU had no significant direct effect on bacterial (p \u3e 0.5) or fungal production (p \u3e 0.3). Extracellular enzyme activities also were not significantly affected by exposure to DCMU. Heterotrophic microbial and enzyme activity assays were conducted in darkness to avoid any indirect effects of DCMU (i.e., heterotrophic responses to the inhibition of photosynthesis, rather than to DCMU itself). The apparent lack of nontarget effects of DCMU on heterotrophic microbial processes, combined with good efficacy against algal photosynthesis, suggest that DCMU may a useful selective inhibitor for investigations of interactions among litter-inhabiting microbiota

Diatom succession in an urban reservoir system

A 43 cm by 5 cm diameter sediment core sample was obtained from Ford Lake reservoir in Washtenaw County, Michigan, and sectioned at 1 cm intervals. The purpose of this study was to determine whether diatom communities in this reservoir have undergone quantifiable changes in abundance and composition since its creation. Thirty-one cm of this core appeared to represent material deposited since the creation of the reservoir based on changes in diatom abundance, the physical composition of the sediment and the change in biogenic SiO 2 concentration. Fortyseven species of diatoms were identified total concentrations of diatom remains varied from 1×10 4 g -1 to 1×10 7 g -1 . Prior to the establishment of the reservoir, the diatom flora was dominated by benthic taxa. Benthic diatoms were numerous throughout the entire core, but eutrophic taxa (e.g., Aulacoseira italica, Aulacoseira granulata, Stephanodiscus niagarae, Fragilaria crotonensis ) dominated much of the core after the reservoir's creation. Total diatom density increased about tenfold in the about the first 10–15 years after the reservoir's creation before declining markedly.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43076/1/10933_2004_Article_BF00213043.pd

Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures