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

Microbial Biotechnolog

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.Microbial Biotechnolog

Repetitive Questioning II

Repetitive questioning is a major problem for caregivers, particularly taxing if they are unable to recognize and understand the reasons why their loved one keeps asking the same question over and over again. Caregivers may be tempted to believe that the patient does not even try to remember the answer given or is just getting obnoxious. This is incorrect. Repetitive questioning is due to the underlying disease: The patient’s short term memory is impaired and he is unable to register, encode, retain and retrieve the answer. If he is concerned about a particular topic, he will keep asking the same question over and over again. To the patient each time she asks the question, it is as if she asked it for the first time. Just answering repetitive questioning by providing repeatedly the same answer is not sufficient. Caregivers should try to identify the underlying cause for this repetitive questioning. In an earlier case study, the patient was concerned about her and her family’s safety and kept asking whether the doors are locked. In this present case study, the patient does not know how to handle the awkward situation he finds himself in. He just does not know what to do. He is not able to adjust to the new unexpected situation. So he repeatedly wants to reassure himself that he is not intruding by asking the same question over and over again. We discuss how the patient’s son-in-law could have avoided this situation and averted the catastrophic ending

Too Many Choices Confuse Patients With Dementia

Choices are often difficult to make by patients with Alzheimer Dementia. They often become acutely confused when faced with too many options because they are not able to retain in their working memory enough information about the various individual choices available. In this case study, we describe how an essentially simple benign task (choosing a dress to wear) can rapidly escalate and result in a catastrophic outcome. We examine what went wrong in the patient/caregiver interaction and how that potentially catastrophic situation could have been avoided or defused

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.Microbial Biotechnolog

The Quantitative Methods Boot Camp:Teaching Quantitative Thinking and Computing Skills to Graduate Students in the Life Sciences

<div><p>The past decade has seen a rapid increase in the ability of biologists to collect large amounts of data. It is therefore vital that research biologists acquire the necessary skills during their training to visualize, analyze, and interpret such data. To begin to meet this need, we have developed a “boot camp” in quantitative methods for biology graduate students at Harvard Medical School. The goal of this short, intensive course is to enable students to use computational tools to visualize and analyze data, to strengthen their computational thinking skills, and to simulate and thus extend their intuition about the behavior of complex biological systems. The boot camp teaches basic programming using biological examples from statistics, image processing, and data analysis. This integrative approach to teaching programming and quantitative reasoning motivates students’ engagement by demonstrating the relevance of these skills to their work in life science laboratories. Students also have the opportunity to analyze their own data or explore a topic of interest in more detail. The class is taught with a mixture of short lectures, Socratic discussion, and in-class exercises. Students spend approximately 40% of their class time working through both short and long problems. A high instructor-to-student ratio allows students to get assistance or additional challenges when needed, thus enhancing the experience for students at all levels of mastery. Data collected from end-of-course surveys from the last five offerings of the course (between 2012 and 2014) show that students report high learning gains and feel that the course prepares them for solving quantitative and computational problems they will encounter in their research. We outline our course here which, together with the course materials freely available online under a Creative Commons License, should help to facilitate similar efforts by others.</p></div

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. Significance Statement Responsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes. Competing Interest Statement The authors have declared no competing interest

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. Significance Statement Responsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes. Competing Interest Statement The authors have declared no competing interest

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.</p

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. Significance Statement Responsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes. Competing Interest Statement The authors have declared no competing interest