Question In Microbiology Research Articles

An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria.

The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

Wiretapping into microbial interactions by single cell genomics.

Wiretapping into microbial interactions by single cell genomics.

Coordinating bacterial cell division with nutrient availability: a role for glycolysis.

ABSTRACTCell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. In this study, we demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacterium Bacillus subtilis. We showed that a deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant and has significant effects on Z-ring formation in wild-type B. subtilis cells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division. Our results support a model in which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown that this protein localizes over the nucleoid in a pyruvate-dependent manner and may stimulate more efficient Z-ring formation at the cell center under nutrient-rich conditions, when cells must divide more frequently.

Crafting 3D Spaces To Take the Ecologic Measure of Microbes

Amid curiosity about microbes in space or on Mars, some experts still wonder how space—more specifically, the three-dimensional (3D) arrangement of one cell to another—affects bacteria on Earth. “That's a big question in microbiology that no one has answered,” says microbiologist Marvin Whiteley. To address that question, he and chemist Jason Shear, both at the University of Texas at Austin, designed 3D microscopic gelatin shells into which they introduced cells of Pseudomonas aeruginosa and Staphylococcus aureus to see whether spatial positioning affects how they behave. It does. In particular, small numbers of P. aeruginosa cells protect larger numbers of S. aureus from being killed by ampicillin. Details appear November 12, 2013 in the Proceedings of the National Academy of Sciences (doi:10.1073/pnas.1309729110).

Nonlinear Spectral Imaging of Fungal Metabolism

Nonlinear microscopy, which in combination with fluorescence spectroscopy is called nonlinear spectral imaging (NLSI), affords access to specimen morphology and (auto)fluorescence spectra at the same time. Thus, it measures the biochemical composition, while also distinguishing different parts of the tissue. Data interpretation is simplified by a new analysis method, the spectral phasor [1], which results in a robust, quick, and semi-blind spectral unmixing of fluorescent species.NLSI and the spectral phasor are a very user-friendly technique and have the potential to address a broad range of microbiological questions. We introduce them as a novel minimum-invasive technique to monitor the state of fungal cells (hyphae). Fungi, both used as consumables and organisms to produce industrial and pharmaceutical compounds, require stringent quality control during their growth. To this end, and as one possible application of NLSI and the spectral phasor, we present their use in monitoring the quality and freshness of white button mushrooms.[1] F. Fereidouni, A.N. Bader, and H.C. Gerritsen, Optics Express, 20,12729-12741 (2012).RGB-representation of a nonlinear spectral image of the white button mushroom (A. bisporus, left) and the corresponding phasor.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Quantification of relative proportions of intact cells in microbiological samples using the example of Cryptosporidium parvum oocysts

The fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm(-2) ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage. Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification.

Animal Models In Mycology: What Have We Learned Over The Past 30 Years

Animal models have long been used to explore various pathophysiological, immunological and microbiological questions in the field of medical mycology. These models have been adapted and altered over time, yet their use has persisted. They remain valuable as research tools due to similarities to processes in human physiology and disease, and are evolving to include more fungal pathogens and infections that better mimic disease in humans. Animal availability, animal cost, housing requirements, the need for immunosuppression, the potential for tissue, fluid or blood samples, a researcher’s familiarity with the model, as well as governmental or institutional regulations, must all be considered when selecting an appropriate one to use. Although the questions of interest have changed over the past 30 years, one idea persists: animal models are valuable tools in research that span the gap between the bench and the clinic.

Transforming clinical microbiology with bacterial genome sequencing

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.

Functional assignment of metagenomic data: challenges and applications.

Metagenomic sequencing provides a unique opportunity to explore earth’s limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely ‘In a given environment, among the microbes present, what are they doing, and how are they doing it?’ Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.

From the regulation of peptidoglycan synthesis to bacterial growth and morphology

How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.

Chemical–Biological Studies of Subcellular Organization in Bacteria

The subcellular organization of biological molecules is a critical determinant of many bacterial processes, including growth, replication of the genome, and division, yet the details of many mechanisms that control intracellular organization remain unknown. Decoding this information will impact the field of bacterial physiology and can provide insight into eukaryotic biology, including related processes in mitochondria and chloroplasts. Small molecule probes provide unique advantages in studying these mechanisms and manipulating the organization of biomolecules in live bacterial cells. In this review, we describe small molecules that are available for investigating subcellular organization in bacteria, specifically targeting FtsZ, MreB, peptidoglycan, and lipid bilayers. We discuss how these probes have been used to study microbiological questions and conclude by providing suggestions about important areas in which chemical-biological approaches will have a revolutionary impact on the study of bacterial physiology.

Introduction of 2,4-dichlorophenoxyacetic acid into soil with solvents and resulting implications for bioavailability to microorganisms

Slow equilibration of introduced chemicals through tortuous pore space limits uniform substrate distribution in soil biodegradation studies. The necessity of introducing poorly soluble xenobiotics via organic solvents, the volume of which is minimized to limit toxicity, likely also affects xenobiotic distribution. Our objective was to investigate relative effects of carrier solvent choice and volume on xenobiotic distribution, apparent solvent toxicity, and soil degradation of 2,4-dichlorophenoxy acetic acid. Incubations using four carrier solvents ranging in properties showed that the fraction of 2,4-D mineralized was a hyperbolic function of solvent volume used (0.02–10 μl g−1), attributed to compensating effects of herbicide bioavailability and solvent toxicity. Substrate concentration influenced mineralization of herbicide introduced with organic carriers, but not water. Fraction of material readily desorbed increased when water was the carrier. Results suggest that solvent toxicity should be balanced with uniformity of substrate distribution when using organic carriers in soils. Substrate bioavailability is a ubiquitous issue in terrestrial microbiology research, thus limitations observed herein broadly apply to microbiology questions about introduced substances in soil. We advocate the development of tools to characterize variable conditions among soil compartments, estimates of substrate bioavailability, and linkage of this information to microbial data.

Chronic <em>Salmonella</em> Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Microbiological aspects of thermally processed foods

The knowledge requirements of the food microbiologist working with thermally processed products are very challenging; particularly in relation to minimally processed foods. In the past, the minimum criteria for sterilized canned foods was very well-defined, however, the diversity of food products, packages and related heat processes are now very difficult to summarize in terms of minimum requirements. The overall heating characteristics of these products can be complex, particularly for mixed particulate products or multicompartment packages. Similarly, the death kinetics of the contaminating micro-organisms can be markedly different dependent on the composition of the food product. These factors combined with the consumer demands for 'fresher' foods has resulted in product, process and package development and innovation progressing at a much faster rate than in the past. While food safety is the most important factor, there are also other issues such as shelf-life and visual quality that have strong market influences and must therefore also be considered by the microbiologist.

A new evaluation of our life-support system.

After more than 150 years of research in microbiology, new technologies and new insights into the microbial world have sparked a revolution in the field. This is a much needed development, not only to renew interest in prokaryote research, but also to meet many emerging challenges in medicine, agriculture and industrial processes. Although many microbiologists—such as Emil von Behring, Robert Koch, Jacques Monod, Francois Jacob, Andre Lwoff, Alexander Fleming, Selman A. Waksman and Joshua Lederberg—grace the list of Nobel laureates, attention moved away from microbiology as biologists focused their interest on eukaryotic cells and higher organisms in the 1970s and 1980s. Furthermore, from the beginning, research on prokaryotes has suffered from an anthropocentric view, regarding as interesting only those organisms that cause disease or that can be exploited for industrial or agricultural use. But the advent of new technologies, some of which have been driven by a need to understand eukaryotes, may change this. We are increasingly realizing how little we know about microbes in general, their diversity, the mechanisms of their evolution and adaptation and their modes of existence within, and communication with, their environment and higher organisms. As bacteria have succeeded in occupying virtually all ecological niches on this planet, ranging from arctic regions to oceanic hot springs, they hold an immense wealth of genetic information that we have barely started to explore and that may provide many useful applications for humans. The new technologies that allow us to sequence and annotate whole genomes more rapidly and to analyse the expression of thousands of genes in a single experiment are likely to speed up this change, particularly as microbes are well suited for high‐throughput analysis. Any microbial genome can now be sequenced within a few hours and, in the near future, new bioinformatics tools will enable scientists not …