Surviving Serum: the Escherichia coli iss Gene of Extraintestinal Pathogenic E. coli Is Required for the Synthesis of Group 4 Capsule.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains constitute a serious and emerging clinical problem, as they cause a variety of infections and are usually highly antibiotic resistant. Many ExPEC strains are capable of evading the bactericidal effects of serum and causing sepsis. One critical factor for the development of septicemia is the increased serum survival (iss) gene, which is highly correlated with complement resistance and lethality. Although it is very important, the function of the iss gene has not been elucidated so far. We have been studying the serum survival of a septicemic strain of E. coli serotype O78, which has a group 4 capsule. Here, we show that the iss gene is required for the synthesis of capsules, which protect the bacteria from the bactericidal effect of complement. Moreover, we show that the deletion of the iss gene results in significantly increased binding of the complement proteins that constitute the membrane attack complex to the bacterial surface.

Extraintestinal Pathogenic Escherichia coli.

Extraintestinal pathogenic E. coli (ExPEC) present a major clinical problem that has emerged in the past years. Most of the infections are hospital or community-acquired and involve patients with a compromised immune system. The infective agents belong to a large number of strains of different serotypes that do not cross react. The seriousness of the infection is due to the fact that most of the infecting bacteria are highly antibiotic resistant. Here, we discuss the bacterial factors responsible for pathogenesis and potential means to combat the infections.

Coping with High Temperature: A Unique Regulation in A. tumefaciens.

Elevation of temperature is a frequent and considerable stress for mesophilic bacteria. Therefore, several molecular mechanisms have evolved to cope with high temperature. We have been studying the response of Agrobacterium tumefaciens to temperature stress, focusing on two aspects: the heat-shock response and the temperature-dependent regulation of methionine biosynthesis. The results indicate that the molecular mechanisms involved in A. tumefaciens control of growth at high temperature are unique and we are still missing important information essential for understanding how these bacteria cope with temperature stress.

Transfer of noncoding DNA drives regulatory rewiring in bacteria.

Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory "switching" to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.

Skin microbiome of people living at the Dead Sea area - The lowest place on earth.

The Dead Sea is a salt lake with surface water at about 430 m below sea level and considered the lowest place on Earth. The Dead Sea basin is characterized by relatively high temperatures, attenuated UV radiation and the air above it has a relatively high-salt aerosol content. When we compared the skin microbiome of individuals from the hot, salty and arid Dead Sea area with that of individuals from the humid Mediterranean regions we observed a significantly lower bacterial diversity in the Dead Sea group as well as distinct differences in the composition of bacterial species. Our results suggest that these factors have a profound effect on the skin microbiome. Further study is required to understand how the local environment influences the skin microbiome, as well as the functional implications of these effects.

Skin microbiome bacteria enriched following long sun exposure can reduce oxidative damage.

Sun exposure is harmful to the skin and increases the risk of skin aging and skin cancer. Here we examined the effects of daily exposure to sun radiation on the skin microbiome in order to determine whether skim microbiome bacteria can contribute to protection from solar damage. Skin swabs were collected from ten lifeguards before and after the summer to analyse the skin microbiome. The results indicate that specific skin microbiome bacteria were enriched following the seasonal sun exposure. Especially interesting were two bacterial families - Sphingomonas and Erythrobacteraceae - which may have the ability to protect against UV radiation as they produce potentially protective compounds. We concentrated on a Sphingomonas strain and could show that it was highly resistant to UV irradiation and was able to reduce reactive oxygen species levels in human keratinocytes. These results provide a proof-of-concept for the role of the skin microbiome in protection from solar radiation.

Insight on Bacterial Newborn Meningitis Using a Neurovascular-Unit-on-a-Chip.

Understanding the pathogenesis of bacterial infections is critical for combatting them. For some infections, animal models are inadequate and functional genomic studies are not possible. One example is bacterial meningitis, a life-threatening infection with high mortality and morbidity. Here, we used the newly developed, physiologically relevant, organ-on-a-chip platform integrating the endothelium with neurons, closely mimicking in vivo conditions. Using high-magnification microscopy, permeability measurements, electrophysiological recordings, and immunofluorescence staining, we studied the dynamic by which the pathogens cross the blood-brain barrier and damage the neurons. Our work opens up possibilities for performing large-scale screens with bacterial mutant libraries for identifying the virulence genes involved in meningitis and determining the role of these genes, including various capsule types, in the infection process. These data are essential for understanding and therapy of bacterial meningitis. Moreover, our system offers possibilities for the study of additional infections-bacterial, fungal, and viral. IMPORTANCE The interactions of newborn meningitis (NBM) with the neurovascular unit are very complex and are hard to study. This work presents a new platform to study NBM in a system that enables monitoring of multicellular interactions and identifies processes that were not observed before.

Host imprints on bacterial genomes--rapid, divergent evolution in individual patients.

Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.

Cellular RNA Targets of Cold Shock Proteins CspC and CspE and Their Importance for Serum Resistance in Septicemic Escherichia coli.

The RNA chaperones, cold shock proteins CspC and CspE, are important in stress response and adaptation. We studied their role in the pathogenesis of a virulent Escherichia coli, representative of extraintestinal pathogenic E. coli (ExPEC) which are serum resistant and septicemic. We performed a global analysis to identify transcripts that interact with these cold shock proteins (CSPs), focusing on virulence-related genes. We used CLIP-seq, which combines UV cross-linking, immunoprecipitation and RNA sequencing. A large number of transcripts bound to the CSPs were identified, and many bind both CspC and CspE. Many transcripts were of genes involved in protein synthesis, transcription and energy metabolism. In addition, there were virulence-related genes, (i.e., fur and ryhB), essential for iron homeostasis. The CLIP-seq results were validated on two transcripts, clpX and tdcA, reported as virulence-associated. Deletion of either CspC or CspE significantly decreased their transcript levels and in a double deletion mutant cspC/cspE, the transcript stability of tdcA and clpX was reduced by 32-fold and 10-fold, respectively. We showed that these two genes are important for virulence, as deleting either of them resulted in loss of serum resistance, a requirement for sepsis. As several virulence-related transcripts interact with CspC or CspE, we determined the importance of these proteins for growth in serum and showed that deletion of either gene significantly reduced serum survival. This phenotype could be partially complemented by cspE and fully complemented by cspC. These results indicate that the two RNA chaperones are essential for virulence, and that CspC particularly critical. IMPORTANCE Virulent Escherichia coli strains that cause infections outside the intestinal tract-extraintestinal pathogenic E. coli (ExPEC)-constitute a major clinical problem worldwide. They are involved in several distinct conditions, including urinary tract infections, newborn meningitis, and sepsis. Due to increasing antibiotic resistance, these strains are a main factor in hospital and community-acquired infections. Because many strains, which do not cross-react immunologically are involved, developing a simple vaccine is not possible. Therefore, it is essential to understand the pathogenesis of these bacteria to identify potential targets for developing drugs or vaccines. One of the least investigated systems involves RNA binding proteins, important for stability of transcripts and global gene regulation. Two such proteins are CspC and CspE ("cold shock proteins"), RNA chaperones involved in stress adaptation. Here we performed a global analysis to identify the transcripts which are affected by these two chaperones, with focus on virulence-associated transcripts.

Effect of Solar Radiation on Skin Microbiome: Study of Two Populations.

Here, we examined the skin microbiome of two groups of healthy volunteers living on the Mediterranean coast with different exposures to sun radiation. One group, exposed to the sun in the summer, was compared with a group covered with clothing throughout the year. The seasonal effects on the skin microbiome of three body sites were determined before and after summer. Surprisingly, at the phyla level, there were no significant differences in microbiome diversity between the groups. Furthermore, within each group, there were no significant seasonal differences in high-abundance species at any of the sampling sites. These results suggest that the skin microbiome, developed over years, remains stable even after several months of exposure to summer weather, direct sunlight and humidity. However, in the group exposed to the sun during the summer months, there were significant differences in low-abundance species in sun-exposed areas of the skin (the inner and outer arm). These subtle changes in low-abundance species are interesting, and their effect on skin physiology should be studied further.

Harnessing Machine Learning To Unravel Protein Degradation in Escherichia coli.

Degradation of intracellular proteins in Gram-negative bacteria regulates various cellular processes and serves as a quality control mechanism by eliminating damaged proteins. To understand what causes the proteolytic machinery of the cell to degrade some proteins while sparing others, we employed a quantitative pulsed-SILAC (stable isotope labeling with amino acids in cell culture) method followed by mass spectrometry analysis to determine the half-lives for the proteome of exponentially growing Escherichia coli, under standard conditions. We developed a likelihood-based statistical test to find actively degraded proteins and identified dozens of fast-degrading novel proteins. Finally, we used structural, physicochemical, and protein-protein interaction network descriptors to train a machine learning classifier to discriminate fast-degrading proteins from the rest of the proteome, achieving an area under the receiver operating characteristic curve (AUC) of 0.72.IMPORTANCE Bacteria use protein degradation to control proliferation, dispose of misfolded proteins, and adapt to physiological and environmental shifts, but the factors that dictate which proteins are prone to degradation are mostly unknown. In this study, we have used a combined computational-experimental approach to explore protein degradation in E. coli We discovered that the proteome of E. coli is composed of three protein populations that are distinct in terms of stability and functionality, and we show that fast-degrading proteins can be identified using a combination of various protein properties. Our findings expand the understanding of protein degradation in bacteria and have implications for protein engineering. Moreover, as rapidly degraded proteins may play an important role in pathogenesis, our findings may help to identify new potential antibacterial drug targets.

Escherichia coli O-antigen capsule (group 4) is essential for serum resistance.

Many septicemic Escherichia coli strains produce polysaccharide capsules, which are important for survival in serum. Here we show that a septicemic E. coli strain of serotype O78 produce an O-antigen capsule (group 4 capsule) and we show that this capsule is essential for serum survival.

Adaptation of Escherichi coli to elevated temperatures involves a change in stability of heat shock gene transcripts.

Bacteria respond to shift-up in temperature by activating the heat shock response - induction of a large number of heat shock genes. This response is essential for adaptation to the higher temperature and for acquiring thermotolerance. One unique feature of the heat shock response is its transient nature - shortly after the induction, the rate of synthesis of heat shock proteins decreases, even if the temperature remains high. Here we show that this shutoff is due to a decrease in the transcript stability of heat shock genes. We further show that the modulation of stability of mRNAs of heat shock genes is maintained by the cold shock protein C - CspC - previously shown to affect transcript stability of specific genes. Upon shifts to higher temperatures the level of this protein decreases due to proteolysis and aggregation, leading to a reduced stability of mRNAs of heat shock genes. The temperature-dependent modulation of transcript stability of heat shock genes constitutes a novel control of the bacterial response to temperature changes.

Involvement of the Pta-AckA pathway in protein folding and aggregation.

Acetyl phosphate is a central metabolite involved in a broad range of versatile cellular functions. Recently it was observed that in Escherichia coli the acetyl phosphate pathway is required for efficient ATP-dependent proteolysis. Deletion of the operon coding for acetyl phosphate metabolism (DeltaackApta) results in a very low cytoplasmic level of acetyl phosphate and impaired proteolysis. Here we show that the DeltaackApta mutation affects additional components of the protein quality control system. Thus, this deletion is accompanied by a decrease in protein refolding and rescue from aggregates. These results indicate the involvement of the acetyl phosphate pathway in chaperone capabilities, in addition to their effect on proteolysis.

Microbial Degradation of Epoxy.

Epoxy resins have a wide range of applications, including in corrosion protection of metals, electronics, structural adhesives, and composites. The consumption of epoxy resins is predicted to keep growing in the coming years. Unfortunately, thermoset resins cannot be recycled, and are typically not biodegradable. Hence, they pose environmental pollution risk. Here, we report degradation of epoxy resin by two bacteria that are capable of using epoxy resin as a sole carbon source. These bacteria were isolated from soil samples collected from areas around an epoxy and polyurethanes manufacturing plant. Using an array of molecular, biochemical, analytical, and microscopic techniques, they were identified as Rhodococcus rhodochrous and Ochrobactrum anthropi. As epoxy was the only carbon source available for these bacteria, their measured growth rate reflected their ability to degrade epoxy resin. Bacterial growth took place only when the two bacteria were grown together, indicating a synergistic effect. The surface morphology of the epoxy droplets changed significantly due to the biodegradation process. The metabolic pathway of epoxy by these two microbes was investigated by liquid chromatography mass spectrometry. Bisphenol A, 3,3'-((propane-2,2-diylbis(4,1-phenylene))bis(oxy))bis(propane-1,2-diol) and some other constituents were identified as being consumed by the bacteria.

Gene expression in Pseudomonas aeruginosa exposed to hydroxyl-radicals.

Recent studies have shown the efficiency of hydroxyl radicals generated via ultraviolet (UV)-based advanced oxidation processes (AOPs) combined with hydrogen peroxide (UV/H2O2) as a treatment process in water. The effects of AOP treatments on bacterial gene expression was examined using Pseudomonas aeruginosa strain PAO1 as a model-organism bacterium. Many bacterial genes are not expressed all the time, but their expression is regulated. The regulation is at the beginning of the gene, in a genetic region called "promoter" and affects the level of transcription (synthesis of messenger RNA) and translation (synthesis of protein). The level of expression of the regulated genes can change as a function of environmental conditions, and they can be expressed more (induced, upregulated) or less (downregulated). Exposure of strain PAO1 to UV/H2O2 treatment resulted in a major change in gene expression, including elevated expression of several genes. One interesting gene is PA3237, which was significantly upregulated under UV/H2O2 as compared to UV or H2O2 treatments alone. The induction of this gene is probably due to formation of radicals, as it is abolished in the presence of the radical scavenger tert-butanol (TBA) and is seen even when the bacteria are added after the treatment (post-treatment exposure). Upregulation of the PA3237 promoter could also be detected using a reporter gene, suggesting the use of such genetic constructs to develop biosensors for monitoring AOPs in water-treatment plants. Currently biosensors for AOPs do not exist, consequently impairing the ability to monitor these processes on-line according to radical exposure in natural waters.

The Escherichia coli Type III Secretion System 2 Has a Global Effect on Cell Surface.

Many strains of Escherichia coli carry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to the Salmonella pathogenicity island 1 (SPI-1) system, encoding a T3SS in Salmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked in E. coli serotype O2-associated with urinary tract infections and septicemia-which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis.IMPORTANCE Drug-resistant extraintestinal pathogenic E. coli (ExPEC) strains are major pathogens, especially in hospital- and community-acquired infections. They are the major cause of urinary tract infections and are often involved in septicemia with high mortality. ExPEC strains are characterized by broad-spectrum antibiotic resistance, and development of a vaccine is not trivial because the ExPEC strains include a large number of serotypes. It is therefore important to understand the virulence factors that are involved in pathogenicity of ExPEC and identify new targets for development of antibacterial drugs or vaccines. Such a target could be ETT2, a unique type III secretion system present (complete or in parts) in many ExPEC strains. Here, we show that this system has a major effect on the bacterial surface-it affects sensitivity to drugs, motility, and secretion of extracellular proteins and outer membrane vesicles. Most importantly, this system is important for serum resistance, a prerequisite for septicemia.

Tools for the study of protein quality control systems: use of truncated homoserine trans-succinylase as a model substrate for ATP-dependent proteolysis in Escherichia coli.

Protein quality control, mediated by chaperones and ATP-dependent proteases, is essential for maintaining balanced growth and for regulating critical processes. To study these systems it is necessary to have model substrate proteins. However, most cellular proteins are stable and the few unstable proteins are usually regulatory and present in low concentrations, making them unsuitable for studies, especially in vivo. We present HTS(Delta1-6), a truncated homoserine trans-succinylase (HTS) which is unstable, can be expressed at high levels and has an enzymatic, measurable, activity. This protein can serve as a good model substrate for Escherichia coli ATP-dependent proteolysis.

Proteomics of septicemic Escherichia coli.

Virulent strains of Escherichia coli have become a major cause of infections, especially in hospitals and institutions, and result in high morbidity and mortality, due to the widespread antibiotic resistance. The infections usually start as complications of urinary tract infections or invasive medical procedures. Septicemic bacteria have to go through the blood stream, where they are exposed to a variety of stress conditions. The most difficult of these is the presence of the immune complement, which is strongly bactericidal. However, recently it has become clear that the nutritional immunity (metabolic stress) of serum is just as important. Thus, as shown by proteomic analyses, septicemic E. coli can cope with this latter stress condition by globally modifying the expression of a variety of metabolic genes. These include genes involved in amino acid metabolism and in metal homeostasis, whose robust regulation of expression appears to be critical for surviving the metabolic immunity of serum. Recognition of the nutritional immunity and the molecular mechanisms that enable septicemic bacteria to overcome it are the focus of this paper.

Evolutionary plasticity of methionine biosynthesis.

Methionine is an essential cellular constituent, the initiator of protein synthesis and a precursor in many metabolic activities, such as methylation and formylation. Here we investigate the genomic distribution of the methionine biosynthetic pathway and analyze its evolutionary history by reconstructing the phylogeny of its enzymatic components. We demonstrate the evolutionary complexity of methionine synthesis and describe the various mechanisms that have shaped this biosynthetic pathway: gene duplication, functional reassignment, lateral acquisition and gene loss. Lateral gene transfer within and between domains and gene recruitment have played an important role in the evolution of this pathway, especially in its first and third enzymatic steps--homoserine activation and homocysteine methylation. These analyses are also the basis of predictions regarding methionine synthesis in Archaea, where the pathway is yet to be characterized. This study illustrates how diverse molecular solutions can fulfill a conserved function in living beings.