Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system.

Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.

Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration.

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

Future increased risk from extratropical windstorms in northern Europe.

European windstorms cause socioeconomic losses due to wind damage. Projections of future losses from such storms are subject to uncertainties from the frequency and tracks of the storms, their intensities and definitions thereof, and socio-economic scenarios. We use two storm severity indices applied to objectively identified extratropical cyclone footprints from a multi-model ensemble of state-of-the-art climate models under different future socio-economic scenarios. Here we show storm frequency increases across northern and central Europe, where the meteorological storm severity index more than doubles. The population-weighted storm severity index more than triples, due to projected population increases. Adapting to the increasing wind speeds using future damage thresholds, the population weighted storm severity index increases are only partially offset, despite a reduction in the meteorological storm severity through adaptation. Through following lower emissions scenarios, the future increase in risk is reduced, with the population-weighted storm severity index increase more than halved.

Defining hierarchical protein interaction networks from spectral analysis of bacterial proteomes.

Cellular behaviors emerge from layers of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be defined from the statistical pattern of proteome variation measured across thousands of diverse bacteria and that these networks reflect the emergence of complex bacterial phenotypes. Our results are validated through gene-set enrichment analysis and comparison to existing experimentally derived databases. We demonstrate the biological utility of our approach by creating a model of motility in Pseudomonas aeruginosa and using it to identify a protein that affects pilus-mediated motility. Our method, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), may be useful for interrogating genotype-phenotype relationships in bacteria.

Pseudomonas aeruginosa AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production.

Pseudomonas aeruginosa employs numerous, complex regulatory elements to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR alleles encoding a phosphomimetic (AlgR D54E) or a phosphoablative (AlgR D54N) form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2 compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration per se AlgR phosphorylation effects were also examined in a Drosophila melanogaster feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated P. aeruginosa virulence in these infection models. These results show that the AlgR phosphorylation state can directly, as well as indirectly, modulate the expression of iron acquisition genes that may ultimately impact the ability of P. aeruginosa to establish and maintain an infection.IMPORTANCE Pyoverdine and pyocyanin production are well-known P. aeruginosa virulence factors that obtain extracellular iron from the environment and from host proteins in different manners. Here, we show that the AlgR phosphorylation state inversely controls pyoverdine and pyocyanin production and that this control is carbon source dependent. P. aeruginosa expressing AlgR D54N, mimicking the constitutively unphosphorylated state, produced more pyocyanin than cells expressing wild-type AlgR. In contrast, a strain expressing an AlgR phosphomimetic (AlgR D54E) produced higher levels of pyoverdine. Pyoverdine production was directly controlled through the prrf2 small regulatory RNA and the pyoverdine sigma factor, PvdS. Abrogating pyoverdine or pyocyanin gene expression has been shown to attenuate virulence in a variety of models. Moreover, the inability to phosphorylate AlgR attenuates virulence in three different models, a Drosophila melanogaster feeding model, a murine acute pneumonia model, and a wound infection model. Interestingly, AlgR-dependent pyoverdine production was responsive to carbon source, indicating that this regulation has additional complexities that merit further study.

The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a multitude of infections. These infections can occur at almost any site in the body and are usually associated with a breach of the innate immune system. One of the prominent sites where P. aeruginosa causes chronic infections is within the lungs of cystic fibrosis patients. P. aeruginosa uses two-component systems that sense environmental changes to differentially express virulence factors that cause both acute and chronic infections. The P. aeruginosa AlgZR two component system is one of its global regulatory systems that affects the organism's fitness in a broad manner. This two-component system is absolutely required for two P. aeruginosa phenotypes: twitching motility and alginate production, indicating its importance in both chronic and acute infections. Additionally, global transcriptome analyses indicate that it regulates the expression of many different genes, including those associated with quorum sensing, type IV pili, type III secretion system, anaerobic metabolism, cyanide and rhamnolipid production. This review examines the complex AlgZR regulatory network, what is known about the structure and function of each protein, and how it relates to the organism's ability to cause infections.

The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus.

Type I interferons (IFN-α and ß) induce dynamic host defense mechanisms to inhibit viral infections. It has been recently recognized that the interferon-inducible transmembrane proteins (IFITM) 1, 2 and 3 can block entry of a broad spectrum of RNA viruses. However, no study to date has focused on the role of IFITM proteins in DNA virus restriction. Here, we demonstrate that IFN-α or -ß treatment of keratinocytes substantially decreases human papillomavirus 16 (HPV16) infection while robustly inducing IFITM1, 2 and 3 expression. However, IFITM1, 2 and 3 overexpression did not inhibit HPV16 infection; rather, IFITM1 and IFITM3 modestly enhanced HPV16 infection in various cell types including primary keratinocytes. Moreover, IFITM1, 2 and 3 did not inhibit infection by two other DNA viruses, human cytomegalovirus (HCMV) and adenovirus type 5 (Ad5). Taken together, we reveal that the entry of several DNA viruses, including HPV, HCMV, and Ad5 is not affected by IFITM1, 2 and 3 expression. These results imply that HPV, and other DNA viruses, may bypass IFITM restriction during intracellular trafficking.