A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630(delta)Derm

The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm. A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm. PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain

First discovery of dolomite and magnesite in living coralline algae and its geobiological implications

Dolomite is a magnesium-rich carbonate mineral abundant in fossil carbonate reef platforms but surprisingly rare in modern sedimentary environments, a conundrum known as the "Dolomite Problem". Marine sedimentary dolomite has been interpreted to form by an unconfirmed, post-depositional diagenetic process, despite minimal experimental success at replicating this. Here we show that dolomite, accompanied by magnesite, forms within living crustose coralline alga, Hydrolithon onkodes, a prolific global tropical reef species. Chemical micro-analysis of the coralline skeleton reveals that not only are the cell walls calcitised, but that cell spaces are typically filled with magnesite, rimmed by dolomite, or both. Mineralogy was confirmed by X-ray Diffraction. Thus there are at least three mineral phases present (magnesium calcite, dolomite and magnesite) rather than one or two (magnesium calcite and brucite) as previously thought. Our results are consistent with dolomite occurrences in coralline algae rich environments in fossil reefs of the last 60 million years. We reveal that the standard method of removing organic material prior to Xray Diffraction analysis can result in a decrease in the most obvious dolomite and magnesite diffraction patterns and this may explain why the abundant protodolomite and magnesite discovered in this study has not previously been recognized. This discovery of dolomite in living coralline algae extends the range of palaeo-environments for which biologically initiated dolomite can be considered a possible source of primary dolomite.M. C. Nash, U. Troitzsch, B. N. Opdyke, J. M. Trafford, B. D. Russell and D. I. Klin

Biomineralization of dolomite and magnesite discovered in tropical coralline algae: a biological solution to the geological dolomite problem

Final revised paper can be found at Description (Link) belowDolomite is a magnesium-rich carbonate mineral abundant in fossil carbonate reef platforms but surprisingly rare in modern sedimentary environments, a conundrum known as the ''Dolomite Problem". Marine sedimentary dolomite has been interpreted to form by an unconfirmed, post-depositional diagenetic process, despite minimal experimental success at replicating this. Here we show that dolomite, accompanied by magnesite, forms within living crustose coralline alga, Hydrolithon onkodes, a prolific global tropical reef species. Chemical micro-analysis of the coralline skeleton reveals that not only are the cell walls calcitised, but that cell spaces are typically filled with magnesite, rimmed by dolomite, or both. Mineralogy was confirmed by X-ray diffraction. Thus there are at least three mineral phases present (magnesium calcite, dolomite and magnesite) rather than one or two (magnesium calcite and brucite) as previously thought. Our results are consistent with dolomite occurrences in coralline algae rich environments in fossil reefs. Instead of a theory of post-depositional dolomitisation, we present evidence revealing biomineralization that can account for the massive formations seen in the geologic record. Additionally, our findings imply that previously unrecognized dolomite and magnesite have formed throughout the Holocene. This discovery together with the scale of coralline algae dominance in past shallow carbonate environments raises the possibility that environmental factors driving this biological dolomitisation process have influenced the global marine magnesium/calcium cycle. Perhaps, most importantly, we reveal that what has been considered a geological process can be a biological process, having many implications for both disciplines.M. C. Nash, U. Troitzsch, B. N. Opdyke, J. M. Trafford, B. D. Russell and D. I. Klinehttp://hdl.handle.net/2440/6855

Muscle Fiber Viability, a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury