Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Use of Liposomes to Study Cellular Osmosensors

When cells are exposed to changes in the osmotic pressure of the external medium, they respond with mechanisms of osmoregulation. An increase of the extracellular osmolality leads to the accumulation of internal solutes by biosynthesis or uptake. Particular bacterial transporters act as osmosensors and respond to increased osmotic pressure by catalyzing uptake of compatible solutes. The functions of osmosensing, osmoregulation , and solute transport of these transporters can be analyzed in molecular detail after solubilization, isolation, and reconstitution into phospholipid vesicles. Using this approach, intrinsic functions of osmosensing transporters are studied in a defined hydrophilic (access to both sides of the membrane) and hydrophobic surrounding (phospholipid membrane), and free of putative interacting cofactors and regulatory proteins

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichia coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress

Integrative analysis of osmoregulation in yeast Saccharomyces cerevisiae

Similar to other unicellular organisms, yeasts frequently encounter environmental stress such as heat shock, osmotic stress, and nutrition limitations, which challenge their growth potential. To survive, all living cells must be able to adapt to changes in their surrounding environment. A set of adaptive responses is triggered that leads to repair of cellular damage in order to overcome these stress conditions. The aim of this thesis is to determine how yeast cells respond to changes in osmolarity and water activity. Upon hyperosmotic shock, water flows out of the cell, resulting in cell shrinkage, and consequently an increase in the concentrations of all substances present in the cytoplasm. Cells adapt their internal osmolarity by gaining an appropriate cell volume as well as an internal water concentration that is optimal for biochemical processes to recover turgor pressure. Osmoregulation is an active process which is mainly regulated by the High Osmolarity Glycerol (HOG) pathway and controls the cellular water balance. The HOG pathway is one of the four yeast MAP kinase pathways. It conveys the hyper osmolarity stress stimulus into the cell machinery and instigates appropriate responses, including global readjustment of gene expression, changes in translational capacity, transient cell cycle arrest, and accumulation of the compatible solute glycerol. Together, these processes result in osmoadaptation. In this thesis I investigated the quantitative characteristics of osmoregulation in the yeast Saccharomyces cerevisiae. I applied a combination of traditional molecular approaches and frontline technologies for comprehensive and quantitative measurements, such as high throughput experiments, synthetic biology, single cell analysis and mathematical modeling to understand the interdependence and timeline of different osmoadaptation process