Versatile selective evolutionary pressure using synthetic defect in universal metabolism

Versatile selective evolutionary pressure using synthetic defect in universal metabolis

Fracture and damage localization in volcanic edifice rocks from El Hierro, Stromboli and Tenerife

© 2018 The Author(s). We present elastic wave velocity and strength data from a suite of three volcanic rocks taken from the volcanic edifices of El Hierro and Tenerife (Canary Islands, Spain), and Stromboli (Aeolian Islands, Italy). These rocks span a range of porosity and are taken from volcanoes that suffer from edifice instability. We measure elastic wave velocities at known incident angles to the generated through-going fault as a function of imposed strain, and examine the effect of the damage zone on P-wave velocity. Such data are important as field measurements of elastic wave tomography are key tools for understanding volcanic regions, yet hidden fractures are likely to have a significant effect on elastic wave velocity. We then use elastic wave velocity evolution to calculate concomitant crack density evolution which ranges from 0 to 0.17: highest values were correlated to the damage zone in rocks with the highest initial porosity

Hydrothermal alteration of andesitic lava domes can lead to explosive volcanic behaviour

Dome-forming volcanoes are among the most hazardous volcanoes on Earth. Magmatic outgassing can be hindered if the permeability of a lava dome is reduced, promoting pore pressure augmentation and explosive behaviour. Laboratory data show that acid-sulphate alteration, common to volcanoes worldwide, can reduce the permeability on the sample lengthscale by up to four orders of magnitude and is the result of pore- and microfracture-filling mineral precipitation. Calculations using these data demonstrate that intense alteration can reduce the equivalent permeability of a dome by two orders of magnitude, which we show using numerical modelling to be sufficient to increase pore pressure. The fragmentation criterion shows that the predicted pore pressure increase is capable of fragmenting the majority of dome-forming materials, thus promoting explosive volcanism. It is crucial that hydrothermal alteration, which develops over months to years, is monitored at dome-forming volcanoes and is incorporated into real-time hazard assessments

Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids

c-di-GMP Turn-Over in Clostridium difficile Is Controlled by a Plethora of Diguanylate Cyclases and Phosphodiesterases

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Abstract Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis

Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack− strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack− and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (−50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia

Detection and Removal of Biases in the Analysis of Next-Generation Sequencing Reads

Since the emergence of next-generation sequencing (NGS) technologies, great effort has been put into the development of tools for analysis of the short reads. In parallel, knowledge is increasing regarding biases inherent in these technologies. Here we discuss four different biases we encountered while analyzing various Illumina datasets. These biases are due to both biological and statistical effects that in particular affect comparisons between different genomic regions. Specifically, we encountered biases pertaining to the distributions of nucleotides across sequencing cycles, to mappability, to contamination of pre-mRNA with mRNA, and to non-uniform hydrolysis of RNA. Most of these biases are not specific to one analyzed dataset, but are present across a variety of datasets and within a variety of genomic contexts. Importantly, some of these biases correlated in a highly significant manner with biological features, including transcript length, gene expression levels, conservation levels, and exon-intron architecture, misleadingly increasing the credibility of results due to them. We also demonstrate the relevance of these biases in the context of analyzing an NGS dataset mapping transcriptionally engaged RNA polymerase II (RNAPII) in the context of exon-intron architecture, and show that elimination of these biases is crucial for avoiding erroneous interpretation of the data. Collectively, our results highlight several important pitfalls, challenges and approaches in the analysis of NGS reads

Biogenesis and functions of bacterial S-layers.

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions