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.

Proteomics of a plant pathogen: Agrobacterium tumefaciens.

Agrobacterium tumefaciens is an important plant pathogen which belongs to the α-proteobacteria. In addition, it has served as the main tool for plant molecular genetics. Here we focus on three major aspects: (i) proteomic mapping, (ii) the use of proteomics for the understanding of the response of A. tumefaciens to changes in environmental conditions and (iii) the analysis of the changes in genome expression following interaction with the host. These studies convey a global outlook on the functional genomics of A. tumefaciens and help to understand the physiology of this important organism.

Biofilm control in water by a UV-based advanced oxidation process.

An ultraviolet (UV)-based advanced oxidation process (AOP), with hydrogen peroxide and medium-pressure (MP) UV light (H(2)O(2)/UV), was used as a pretreatment strategy for biofilm control in water. Suspended Pseudomonas aeruginosa cells were exposed to UV-based AOP treatment, and the adherent biofilm formed by the surviving cells was monitored. Control experiments using H(2)O(2) or MP UV irradiation alone could inhibit biofilm formation for only short periods of time (<24 h) post-treatment. In a H(2)O(2)/filtered-UV (>295 nm) system, an additive effect on biofilm control was shown vs filtered-UV irradiation alone, probably due to activity of the added hydroxyl radical (OH•). In a H(2)O(2)/full-UV (ie full UV spectrum, not filtered) system, this result was not obtained, possibly due to the germicidal UV photons overwhelming the AOP system. Generally, however, H(2)O(2)/UV prevented biofilm formation for longer periods (days) only when maintained with residual H(2)O(2). The ratio of surviving bacterial concentration post-treatment to residual H(2)O(2) concentration played an important role in biofilm prevention and bacterial regrowth. H(2)O(2) treatments alone resulted in poorer biofilm control compared to UV-based AOP treatments maintained with similar levels of residual H(2)O(2), indicating a possible advantage of AOP.

Biofilm control in water by advanced oxidation process (AOP) pre-treatment: effect of natural organic matter (NOM).

The main goal of this study was to examine the influence of natural organic matter (NOM) on the efficiency of H2O2/UV advanced oxidation process (AOP) as a preventive treatment for biofilm control. Pseudomonas aeruginosa PAO1 biofilm-forming bacteria were suspended in water and exposed to various AOP conditions with different NOM concentrations, and compared to natural waters. H2O2/UV prevented biofilm formation: (a) up to 24 h post treatment - when residual H2O2 was neutralized; (b) completely (days) - when residual H2O2 was maintained. At high NOM concentrations (i.e. 25 mg/L NOM or 12.5 mg/L DOC) an additive biofilm control effect was observed for the combined H2O2/UV system compared to UV irradiation alone, after short biofilm incubation times (<24 h). This effect was H2O2 concentration dependent and can be explained by the high organic content of these water samples, whereby an increase in NOM could enhance (•)OH production and promote the formation of additional reactive oxygen species. In addition, maintaining an appropriate ratio of bacterial surviving conc.: residual H2O2 conc. post-treatment could prevent bacterial regrowth and biofilm formation.

The heat shock protein YbeY is required for optimal activity of the 30S ribosomal subunit.

The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.

Distribution and evolution of virulence factors in septicemic Escherichia coli.

Bacterial septicemia is an emerging clinical problem which is increasing in significance due to the rapid spread of antibiotic resistance. In order to combat this problem, it is essential to identify the critical virulence factors of these septicemic strains and study their functions and role in pathogenesis. Here, we survey some of the virulence factors which are essential for sepsis and are potential candidates for development of new drugs or vaccines.

Biofouling control in water by various UVC wavelengths and doses.

UV light irradiation is being increasingly applied as a primary process for water disinfection, effectively used for inactivation of suspended (planktonic) cells. In this study, the use of UV irradiation was evaluated as a pretreatment strategy to control biofouling. The objective of this research was to elucidate the relative effectiveness of various targeted UV wavelengths and a polychromatic spectrum on bacterial inactivation and biofilm control. In a model system using Pseudomonas aeruginosa, the inactivation spectra corresponded to the DNA absorption spectra for all wavelengths between 220 and 280 nm, while wavelengths between 254 nm and 270 nm were the most effective for bacterial inactivation. Similar wavelengths of 254-260-270 nm were also more effective for biofilm control in most cases than targeted 239 and 280 nm. In addition, the prevention of biofilm formation by P. aeruginosa with a full polychromatic lamp was UV dose-dependent. It appears that biofilm control is improved when larger UV doses are given, while higher levels of inactivation are obtained when using a full polychromatic MP lamp. However, no significant differences were found between biofilms produced by bacteria that survived UV irradiation and biofilms produced by control bacteria at the same microbial counts. Moreover, the experiments showed that biofilm prevention depends on the post-treatment incubation time and nutrient availability, in addition to targeted wavelengths, UV spectrum and UV dose.

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.

Development of vaccines at the time of COVID-19.

In December 2019, a working group of the European Academy of Microbiology assembled to discuss various aspects of vaccines and vaccinations. The meeting was organised by Jörg Hacker and Eliora Z. Ron and took place in the offices of the Leopoldina (German National Academy of Sciences Leopoldina). Several important issues were addressed and a major part of the discussion focused on the need to develop new vaccines, especially to protect against pathogens that constitute a pandemic threat. Following the rapid and unpredicted spread of COVID-19 in the first seven months of 2020, the need to develop vaccines for pandemic viruses rapidly has been clearly established. Thus, this paper will concentrate on points that were highlighted by the recent COVID-19 pandemic and lessons learnt therefrom.

YbeY, a heat shock protein involved in translation in Escherichia coli.

Here we provide evidence that YbeY, a conserved heat shock protein with unknown function, is involved in the translation process. ybeY deletion mutants are temperature sensitive and have a significantly reduced thermotolerance. Nonetheless, there appears to be no damage of the protein quality control of mature polypeptides, as the levels of chaperones and proteases are normal and there is no accumulation of aggregates. Rather, the mutation results in a significant reduction in the level of polysomes, and upon a shift to a restrictive temperature (42 degrees C), there is an immediate and severe slowdown of translation. Taken together, the data indicate that YbeY is an important factor for bacterial translation even at 37 degrees C but becomes essential at high temperatures.

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.