Study on late competence proteins involved in natural transformation of Bacillus subtilis

The following study comprises in vivo and in vitro data on several of the so-called late competence proteins, which are involved in natural transformation of Bacillus subtilis. The gram-positive bacterium B. subtilis belongs to those bacteria, who are able to take up DNA from their environment and incorporate the foreign DNA by homologous recombination into their own chromosome; a feature named competence. This fascinating ability is carried out by only a portion of the bacterial culture, expressing specific proteins, encoded by the late competence operons. If exogenous double-stranded DNA is about to be taken up from the environment, it needs to first cross the thick cell wall of B. subtilis, with a width of ~40 nm. In case of B. subtilis, this first border is crossed by a putative pseudopilus who transfers the DNA inside of the cell. The energy for this particular process is probably provided by the assembly/disassembly ATPase ComGA. The taken-up DNA is then further transferred into the cytosol by the so-called competence complex or competence machinery. The complex consists out of specific competence proteins, which assemble at the membrane, including a DNA-binding transmembrane protein (ComEA) and an aqueous channel protein (ComEC). In the following thesis, the unknown role of the protein ComEB has been further elucidated in the context of competence, and its enzymatic function was analysed in vitro. It was found that the protein carries out deaminase activity, which is not essential for transformation. In case of ComEC, an amino acid, D573, has been identified as essential for transformation. Truncations of the protein, supposed to carry out an exonuclease function, were heterologously expressed and purified as GST-tag fusions, but, probably due to aggregations of the proteins, no enzymatic activity was detected. The intracellular diffusion of fluorophore fusions of several competence proteins, namely ComEB-mV, ComGA-mV, ComEC-mV and mV-ComEA was analysed via single-molecule tracking, in the presence and absence of exogeneous DNA. In case of ComGA, a C-terminal fusion to mVenus was analysed and it was found that the protein becomes more dynamic in the presence of DNA. Further, the localization and diffusion of a fluorescently labeled PCR product inside of competent Bacillus cells was analysed for the first time. The diffusive behaviour and localization of the stained DNA resembles the diffusion of mV-ComEA. This led to the hypothesis that ComEA serves as a reservoir for taken-up DNA, similar to what is already known for orthologues of ComEA from other naturally competent bacteria

<scp>ReSurveyEurope</scp>: A database of resurveyed vegetation plots in Europe

AbstractAimsWe introduce ReSurveyEurope — a new data source of resurveyed vegetation plots in Europe, compiled by a collaborative network of vegetation scientists. We describe the scope of this initiative, provide an overview of currently available data, governance, data contribution rules, and accessibility. In addition, we outline further steps, including potential research questions.ResultsReSurveyEurope includes resurveyed vegetation plots from all habitats. Version 1.0 of ReSurveyEurope contains 283,135 observations (i.e., individual surveys of each plot) from 79,190 plots sampled in 449 independent resurvey projects. Of these, 62,139 (78%) are permanent plots, that is, marked in situ, or located with GPS, which allow for high spatial accuracy in resurvey. The remaining 17,051 (22%) plots are from studies in which plots from the initial survey could not be exactly relocated. Four data sets, which together account for 28,470 (36%) plots, provide only presence/absence information on plant species, while the remaining 50,720 (64%) plots contain abundance information (e.g., percentage cover or cover–abundance classes such as variants of the Braun‐Blanquet scale). The oldest plots were sampled in 1911 in the Swiss Alps, while most plots were sampled between 1950 and 2020.ConclusionsReSurveyEurope is a new resource to address a wide range of research questions on fine‐scale changes in European vegetation. The initiative is devoted to an inclusive and transparent governance and data usage approach, based on slightly adapted rules of the well‐established European Vegetation Archive (EVA). ReSurveyEurope data are ready for use, and proposals for analyses of the data set can be submitted at any time to the coordinators. Still, further data contributions are highly welcome.</jats:sec

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia.

MAIN CONCLUSION Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites

Single molecule tracking of ComGC^CYS with Alexa-Fluor C5-maleimide treatment, with/ and without addition of DNA with an incubation time of 10 minutes.

Single molecule tracking of ComGCCYS with Alexa-Fluor C5-maleimide treatment, with/ and without addition of DNA with an incubation time of 10 minutes.</p

Time lapse (20 second intervals) of pilus structures of AF488-C5 maleimide stained <i>B</i>. <i>subtilis</i> cells grown to competence expressing ComGC^CYS.

Time lapse (20 second intervals) of pilus structures of AF488-C5 maleimide stained B. subtilis cells grown to competence expressing ComGCCYS.</p

Reconstruction of a Z-stacks of an AF488-C5 maleimide stained <i>B</i>. <i>subtilis</i> cell grown to competence expressing ComGC^CYS.

Reconstruction of a Z-stacks of an AF488-C5 maleimide stained B. subtilis cell grown to competence expressing ComGCCYS.</p

Single molecule tracking of ComGC and ComGC-CYS strains with and without treatment with AF488-C5 maleimide.

Confinement maps of A) PY79, amyE::comGC with maleimide (A) and without (B), and PY79, amyE::comGCCYS with (D) and without (C) treatment of AF488-C5 maleimide. Confinement radius 108.7 nm (E) Bubble plots show the different sizes for populations of ComGC and ComGC-CYS in cells treated with (+) and without (-) stain. Size of bubbles indicates the size of populations. (TIF)</p

Epifluorescence microscopy of AF488 C5-maleimide stained cells of mutated cells (PY79 <i>amyE</i>::<i>comGC</i>^<i>CYS</i><i>)</i> and cells (PY79 <i>amyE</i>::<i>comGC)</i> encoding ComGC at the <i>amyE</i> locus.

With 0.5 mM IPTG induction (panel A and B), without IPTG induction (panel C) and with low (0.01 mM) concentrations of IPTG (panel D). Left images of a panel show bright field images (BF-channel), right panels show epifluorescence pictures (GFP-channel, i.e. imaging of 488 nm fluorescence excitation). Scale bars represent 2 μm.</p

Response of pilus dynamics after addition of DNA.

Panels A) and B) show heat maps of confined tracks of (AF488 C5-maleimide labelled) ComGCCYS in cells projected into a standardized B. subtilis cell. Confinement radius was 108.7 nm. Colour code on the right indicates intensity of signals. “- DNA” and “+ DNA” indicates untreated cells or cells treated with DNA for 10 minutes. C) Bubble plot shows diffusion constants [μm2/s] the fraction sizes of populations by setting a simultaneous diffusion constant which is shown in correspondence. Errors correspond to the 95% confidence intervals which are given by “confint” matlab function by using its values that result from the fit. +DNA and -DNA indicates cells which were treated with DNA for 10 minutes.</p