The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae.

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.

An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

An Open-Source Storage Solution for Cryo-Electron Microscopy Samples.

Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.

How advances in cryo-electron tomography have contributed to our current view of bacterial cell biology.

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

UVC inactivation of pathogenic samples suitable for cryo-EM analysis.

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

The frequency of inherited hydrocephalus is influenced by intrauterine factors in H-Tx rats.

H-Tx rats have fetal-onset inherited hydrocephalus. Linkage analysis has determined the genetics is complex, with at least three loci associated with hydrocephalus. In addition, maternal and/or intrauterine factors influence the frequency of expression. The aim of this study was to characterize nongenetic (epigenetic) factors that affect hydrocephalus in this strain. Groups of primiparous and multiparous females were used to breed fetuses for examination in utero. Multiparous females were manipulated to have either gestation with lactation or, by removal of pups at birth, gestation without lactation. In addition, hydrocephalus expression in postnatal rats from the breeding colony was analyzed for primiparous and multiparous females. The latter were subdivided according to the interval between the litter examined and the previous litter. There was no particular uterine position or horn that favored hydrocephalus and hydrocephalic fetuses were the same weight as normal littermates. The frequency of hydrocephalus was 16-20% in primiparous females and twofold higher in multiparous females that were lactating during pregnancy. Removal of the suckling pups prevented this increase. The severity of hydrocephalus was measured on 1-mm-thick fixed brain slices. Fetuses from lactating females had hydrocephalus that was significantly more severe than the nonlactating groups. However, all fetus groups had hydrocephalus that was very much less severe than the postnatal pups, suggesting that severity increases after birth. It is concluded that there is an epigenetic factor that increases the frequency of inherited hydrocephalus in fetuses if suckling pups are present during gestation. Future experiments will examine possible mechanisms for this genotype-environment interaction.