Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established, however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used EMS mutagenesis and a motility assay to identify motile suppressors of the ΔpilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. Overexpression of these arlI and arlJ mutant constructs in the respective multi-deletion strains ΔpilA[1-6]ΔarlI and ΔpilA[1-6]ΔarlJ confirmed their role in suppressing the ΔpilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA overexpression in wild-type cells led to decreased motility. Moreover, qRT-PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ΔcirA cells, which form rods during both early and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.

Microbiology Laboratory Simulations: From a Last-Minute Resource during the Covid-19 Pandemic to a Valuable Learning Tool to Retain-A Semester Microbiology Laboratory Curriculum That Uses Labster as Prelaboratory Activity.

As educators, we strive to provide the best delivery method to improve our students' learning experience. The Covid-19 shutdown posed an incredible challenge by forcing us to redesign the way we teach with no time to prepare. Bringing the laboratory curriculum outside the lab was one of the most challenging tasks. Instructors got creative, adopting safe, at-home laboratory experiments, at-home kits bought from distributors, and relying on online simulations. Now that we are returning to face-to-face meetings, the experience gained during the lockdown can be harnessed and used as extra tools available for our students' learning experience. Online simulations gained attention during the lockdown and turned out to be of great value, with different studies reporting on improved students' experiences following the use of simulations. This paper outlines how to incorporate Labster simulations in an in-person microbiology semester for undergraduate students.

A Twist to the Kirby-Bauer Disk Diffusion Susceptibility Test: an Accessible Laboratory Experiment Comparing Haloferax volcanii and Escherichia coli Antibiotic Susceptibility to Highlight the Unique Cell Biology of Archaea.

Archaea, once thought to only live in extreme environments, are present in many ecosystems, including the human microbiome, and they play important roles ranging from nutrient cycling to bioremediation. Yet this domain is often overlooked in microbiology classes and rarely included in laboratory exercises. Excluding archaea from high school and undergraduate curricula prevents students from learning the uniqueness and importance of this domain. Here, we have modified a familiar and popular microbiology experiment-the Kirby-Bauer disk diffusion antibiotic susceptibility test-to include, together with the model bacterium Escherichia coli, the model archaeon Haloferax volcanii. Students will learn the differences and similarities between archaea and bacteria by using antibiotics that target, for example, the bacterial peptidoglycan cell wall or the ribosome. Furthermore, the experiment provides a platform to reiterate basic cellular biology concepts that students may have previously discussed. We have developed two versions of this experiment, one designed for an undergraduate laboratory curriculum and the second, limited to H. volcanii, that high school students can perform in their classrooms. This nonpathogenic halophile can be cultured aerobically at ambient temperature in high-salt media, preventing contamination, making the experiment low-cost and safe for use in the high school setting.

Undergraduate Research in the Time of COVID-19: A Remote Imaging Protocol for Physically Distanced Students Studying Wildlife.

The COVID-19 pandemic has shuttered many university research labs because campuses are closed, and faculty and students lack productive ways of working remotely. This presents major difficulties for students who need research opportunities to fulfill their intellectual growth potential and their undergraduate research and thesis requirements. Without research experiences, undergraduates may be less competitive for future jobs and graduate programs. Similarly, faculty need research avenues to advance their academic careers while maintaining physically distant protocols. We outline here a budget-friendly, COVID-friendly, adaptable protocol that aims to introduce students to the wildlife research opportunities surrounding their campus or home through observation and literature research. Student researchers learn the scientific method by getting first-hand experience with an original research project. The pedagogical goals include designing a study: defining a question or proposing a hypothesis, collecting, organizing, and analyzing data, and sharing results in the form of posters, theses, informal educational materials, and scientific publications. This protocol is flexible to allow for different budgets, opportunities, and constraints. The researchers monitor different locations using trail cameras to determine which species are present around campus or even students' homes. During the COVID-19 pandemic, when it is likely there will be few in-person meetings, this protocol offers students the opportunity to carry out research with limited or no in-person meetings, and it can be run remotely by sharing the data collected. In this paper, we provide instructions, details, and student handouts for instructors to help implement this research project.

Exposure and post-exposure effects of chlorpyrifos on Carassius auratus gills: An ultrastructural and morphofunctional investigation.

Widespread environmental contamination from chlorpyrifos (CPF) is well acknowledged and has led to the proposal to ban or limit its use in agricultural and domestic, within the regulatory context of both America and Europe. Furthermore, great concerns arise as to whether exposure to CPF represents a potential risk to human health. In the present study, by subjecting the goldfish model to three environmentally realistic concentrations of CPF (1, 4, and 8 µg/L) for 96 h, we demonstrated that this pesticide has the potential to induce severe morphological, ultrastructural and functional alterations in gills, even at very low concentrations. The degree of pathological effects was dose-dependent, and the main morphological alterations recorded were: regression of interlamellar cellular mass (ILCM), hypertrophy, and hyperplasia of epithelial cells, degeneration of both chloride cells and pillar cells. CPF exposure resulted in a decrease of Na+/K+-ATPase expression and the induction of iNOS, as revealed by immunohistochemical analysis. In order to determine the overall toxicity of CPF, we also investigated the recovery capability of goldfish gills following a period of 7 days in pesticide-free water. Our results clearly showed that there exists a threshold of CPF dose below which the effects on gills are reversible and beyond which the ability of gills to recover their typical features is completely lost. The information presented in this paper emphasises the importance of evaluating the recovery ability of organisms after chemical input and enhances our knowledge of the potential hazard of organophosphorus pesticides (OPs) on freshwater ecosystems.

Lead toxicity in seawater teleosts: A morphofunctional and ultrastructural study on the gills of the Ornate wrasse (Thalassoma pavo L.).

Lead is one of the most alarming toxic pollutants for the environment due to its acute toxicity and high bioaccumulation tendency. Despite legislative efforts, world lead production has more than doubled since the early 1970s to 2016. Due to extensive exploitation and human activity, the coastal and estuarine regions belong to marine environments that are mostly and more rapidly deteriorated by pollutants including lead. A limited number of studies examined the effects of lead in fishes, compared to other aquatic models and even fewer studies have been dedicated to seawater fishes especially regarding Pb adsorption and accumulation in specific organs. Fish gills, key compartments involved in several crucial functions such as gas exchange, osmoregulation, and excretion, are also the organs in which Pb is mainly accumulated. Herein, we investigated the morphofunctional and ultrastructural modifications in the gills of a marine teleost (Thalassoma pavo) after acute exposure (48, 96, 192 h) to three lead concentrations. We showed that pathological alterations can be detected in all the examined samples. The most common modifications observed were: the curling of the lamellae and the dilation of their apical tips, the lamellar edema, the proliferation and the hypertrophy of CCs, the progressive epithelial disorganization with detachment of the epithelium from connective tissue. This study also demonstrates that there is a weak influence on the expression pattern of Na+/K+-ATPase and AQP3 biomarker enzymes while high metallothioneins expression has been observed. The described alterations may adversely affect gas exchange and ionic balance, with a long chain of cascading effects. This is the first evidence of the effects exerted by lead on gills of seawater fishes that highlights the harmful properties of this metal, even at low concentration.

Morphological and Immunohistochemical Modifications in Zebrafish (Danio rerio) Gills After Short-Term Exposure to the Fungicide Tebuconazole.

In agroecosystems, a variety of pesticides may enter aquatic habitats exerting a number of toxic effects on living organisms. Due to their wide use and the repeated application, fungicides can be found at a much greater concentration than other agrochemicals in all environmental compartments. One of these fungicides, tebuconazole (TBZ), has been recognized as an endocrine-disrupting chemical, and the available literature focuses mainly on this mechanism of action. In the present study by subjecting the zebrafish model to an environmentally realistic concentration of TBZ, we clearly demonstrate that this fungicide has the potential to induce severe morphological and ultrastructural alterations in gills. The main morphological effects recorded in this study were as follows: the proliferation of the main epithelium, ectopia of chloride cells, lamellar shortening, necrosis, and apoptosis. TBZ exposure also resulted in the overtime decrease in the expression of both Na+/K+-ATPase and aquaporin 3 and in the induction of oxidative stress enzyme (superoxide dismutase-1) as revealed by immunohistochemical analysis. Our results clearly indicate that respiratory and osmoregulatory disorders represent an important mechanism of TBZ toxicity on Danio rerio gills. This is the first evidence of sublethal effects exerted by TBZ on fish gills and highlights the harmful properties of this fungicide, even at very low concentration.

Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

Effects of salinity stress on Bufo balearicus and Bufo bufo tadpoles: Tolerance, morphological gill alterations and Na(+)/K(+)-ATPase localization.

Freshwater habitats are globally threatened by human-induced secondary salinization. Amphibians are generally poorly adapted to survive in saline environments. We experimentally investigated the effects of chronic exposure to various salinities (5%, 10%, 15%, 20%, 25%, 30% and 35% seawater, SW) on survival, larval growth and metamorphosis of tadpoles from two amphibian populations belonging to two species: the green toad Bufo balearicus and the common toad Bufo bufo. In addition, gill morphology of tadpoles of both species after acute exposure to hypertonic conditions (20%, 25%, and 30% SW) was examined by light and electron microscopy. Tadpoles experienced 100% mortality above 20% SW in B. balearicus while above 15% SW in B. bufo. We detected also sublethal effects of salinity stress on growth and metamorphosis. B. bufo cannot withstand chronic exposure to salinity above 5% SW, tadpoles grew slower and were significantly smaller than those in control at metamorphosis. B. balearicus tolerated salinity up to 20% SW without apparent effects during larval development, but starting from 15% SW tadpoles metamorphosed later and at a smaller size compared with control. We also revealed a negative relation between increasing salt concentration and gill integrity. The main modifications were increased mucous secretion, detachment of external layer, alteration of epithelial surface, degeneration phenomena, appearance of residual bodies, and macrophage immigration. These morphological alterations of gill epithelium can interfere with respiratory function and both osmotic and acid-base regulation. Significant variations in branchial Na(+)/K(+)-ATPase activity were also observed between two species; moreover an increase in enzyme activity was evident in response to SW exposure. Epithelial responses to increasing salt concentration were different in the populations belonging to two species: the intensity of histological and ultrastructural pathology in B. bufo was greater and we noticed the appearance in exposed samples of the tubular vesicle cells (TVCs). Taken together, our results demonstrated that increased salinity of freshwater may give cause for concern and must be considered a stressor for amphibians as well as other pollutants.

N-glycosylation of Haloferax volcanii flagellins requires known Agl proteins and is essential for biosynthesis of stable flagella.

N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

Archaeal type IV pilus-like structures--evolutionarily conserved prokaryotic surface organelles.

In both bacteria and Archaea, the biosynthesis of type IV pilus-related structures involves a set of core components, including a prepilin peptidase that specifically processes precursors of pilin-like proteins. Although in silico analyses showed that most sequenced archaeal genomes encode predicted pilins and conserved pilus biosynthesis components, recent in vivo analyses of archaeal pili in genetically tractable crenarchaea and euryarchaea revealed Archaea-specific type IV pilus functions and biosynthesis components. Studies in a variety of archaeal species will reveal which type IV pilus-like structures are common in Archaea and which are limited to certain species within this domain. The insights gleaned from these studies may also elucidate the roles played by these types of structures in adapting to specific environments.

Haloferax volcanii flagella are required for motility but are not involved in PibD-dependent surface adhesion.

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.

Dehydration-induced expression of a 31-kDa dehydrin in Polypodium polypodioides (Polypodiaceae) may enable large, reversible deformation of cell walls.

Current and predicted climate changes caused by global warming compel greater understanding of the molecular mechanisms that plants use to survive drought. The desiccation-tolerant fern Polypodium polypodioides exhibits extensive cell wall folding when dried to less than 15% relative water content (RWC) and rapidly (within 24 h) rehydrates when exposed to water and high humidity. A 31-kDa putative dehydrin polypeptide expressed in partially and fully dry tissues detected via western blotting was present only during drying and rapidly dissipated (within 24 h) upon tissue rehydration. Immunostaining indicates the presence of dehydrin near the cell wall of partially and fully dried tissues. Atomic force microscopy of tracheal scalariform perforations indicates that dry vascular tissue does not undergo significant strain. Additionally, environmental scanning electron microscopy reveals differential hydrophilicity between the abaxial and adaxial leaf surfaces as well as large, reversible deformation. The ability to avoid cell wall damage in some desiccation-tolerant species may be partially attributed to cell wall localization of dehydrins enabling reversible, large cell-wall deformation. Thus, the de novo synthesis of dehydrin proteins and potential localization to the cell walls of these desiccation-tolerant species may play a role in avoiding mechanical failure during drought.