Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.

Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17-deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week -3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.

The evolution of small-colony variants within Pseudomonas aeruginosa populations chronically infecting the cystic fibrosis lung is one example of the emergence of adapted subpopulations. Here, we present the complete genome sequence of the autoaggregative and hyperpiliated P. aeruginosa small-colony variant SCV20265, which was isolated from a cystic fibrosis (CF) patient.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

The opportunistic human pathogen Pseudomonas aeruginosa can thrive under microaerophilic to anaerobic conditions in the lungs of cystic fibrosis patients. RNASeq based comparative RNA profiling of the clinical isolate PA14 cultured in synthetic cystic fibrosis medium was performed after planktonic growth (OD600 = 2.0; P), 30 min after shift to anaerobiosis (A-30) and after anaerobic biofilm growth for 96h (B-96) with the aim to reveal differentially regulated functions impacting on sustained anoxic biofilm formation as well as on tolerance towards different antibiotics. Most notably, functions involved in sulfur metabolism were found to be up-regulated in B-96 cells when compared to A-30 cells. Based on the transcriptome studies a set of transposon mutants were screened, which revealed novel functions involved in anoxic biofilm growth.In addition, these studies revealed a decreased and an increased abundance of the oprD and the mexCD-oprJ operon transcripts, respectively, in B-96 cells, which may explain their increased tolerance towards meropenem and to antibiotics that are expelled by the MexCD-OprD efflux pump. The OprI protein has been implicated as a target for cationic antimicrobial peptides, such as SMAP-29. The transcriptome and subsequent Northern-blot analyses showed that the abundance of the oprI transcript encoding the OprI protein is strongly decreased in B-96 cells. However, follow up studies revealed that the susceptibility of a constructed PA14ΔoprI mutant towards SMAP-29 was indistinguishable from the parental wild-type strain, which questions OprI as a target for this antimicrobial peptide in strain PA14.

Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.

Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine.

The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

Most bacteria control oxidative stress through the H(2)O(2)-responsive transactivator OxyR, a member of the LTTR family (LysR Type Transcriptional Regulators), which activates the expression of defensive genes such as those encoding catalases, alkyl hydroperoxide reductases and superoxide dismutases. In the human opportunistic pathogen Pseudomonas aeruginosa, OxyR positively regulates expression of the oxidative stress response genes katA, katB, ahpB and ahpCF. To identify additional targets of OxyR in P. aeruginosa PAO1, we performed chromatin immunoprecipitation in combination with whole genome tiling array analyses (ChIP-chip). We detected 56 genes including all the previously identified defensive genes and a battery of novel direct targets of OxyR. Electrophoretic mobility shift assays (EMSAs) for selected newly identified targets indicated that ∼70% of those were bound by purified oxidized OxyR and their regulation was confirmed by quantitative real-time polymerase chain reaction. Furthermore, a thioredoxin system was identified to enzymatically reduce OxyR under oxidative stress. Functional classification analysis showed that OxyR controls a core regulon of oxidative stress defensive genes, and other genes involved in regulation of iron homeostasis (pvdS), quorum-sensing (rsaL), protein synthesis (rpsL) and oxidative phosphorylation (cyoA and snr1). Collectively, our results indicate that OxyR is involved in oxidative stress defense and regulates other aspects of cellular metabolism as well.

Peroxisome proliferator-activated receptor delta belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. It is a key regulator of lipid metabolism. The peroxisome proliferator-activated receptor delta gene (PPARD) has been assigned to a region on porcine chromosome 7, which harbours a quantitative trait locus for backfat. Thus, PPARD is considered a functional and positional candidate gene for backfat thickness. The purpose of this study was to test this candidate gene hypothesis in a cross of breeds that were highly divergent in lipid deposition characteristics.

Telomeres are short and repetitive sequences at the ends of linear chromosomes which shorten with every cell-division in vitro. Telomere length (TL) is reported to decrease with age and stress. The domesticated water buffalo (Bubalus bubalis) is the second most important milk producing animal worldwide. The productive lifespan of water buffalo cows is reported to be longer than that of dairy cows (Bos taurus). With this background, we aimed to compare TL in leukocytes obtained from blood samples from water buffaloes across different ages. In addition, we tested the suitability of assessing TL in DNA derived from nasal and vaginal epithelial cells via swabs as potential non-invasive alternatives to blood sampling Samples were collected from 20 calves (3 months of age), 20 heifers (2 years old), 20 cows (1st lactation, 3 years old), and 13 cows (3rd lactation, about 5 years old). We found that TL in leukocytes from water buffalo calves, heifers, and from cows in their first lactation was not different, but shorter telomeres were observed in cows in their third lactation. The results thus support an age-dependent decrease of TL in water buffaloes. Leukocyte TL was weakly correlated with TL measured in DNA from nasal epithelial cells (r = 0.327; P = .025), but not with TL from vaginal epithelial cells. Due to the poor correlation between epithelial cell and leukocyte TL and to the difficulties with collecting nasal swabs, we conclude that they are no suitable alternatives to blood samples for telomere studies in water buffaloes.

Previous studies have demonstrated substantial genetic diversification of Pseudomonas aeruginosa across sub-compartments in cystic fibrosis (CF) lungs. Here, we isolate P. aeruginosa from five different sampling areas in the upper and lower airways of an explanted CF lung, analyze ex vivo transcriptional profiles by RNA-seq, and use colony re-sequencing and deep population sequencing to determine the genetic diversity within and across the various sub-compartments. We find that, despite genetic variation, the ex vivo transcriptional profiles of P. aeruginosa populations inhabiting different regions of the CF lung are similar. Although we cannot estimate the extent to which the transcriptional response recorded here actually reflects the in vivo transcriptomes, our results indicate that there may be a common in vivo transcriptional profile in the CF lung environment.

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.

Pseudomonas aeruginosa is an environmental bacterium and an opportunistic human pathogen. It is also a well-established model organism to study bacterial adaptation to stressful conditions, such as those encountered during an infection process in the human host. Advancing knowledge on P. aeruginosa adaptation to biofilm growth conditions is bound to reveal novel strategies and targets for the treatment of chronic biofilm-associated infections. Here, we generated transposon insertion libraries in three P. aeruginosa strain backgrounds and determined the relative frequency of each insertion following biofilm growth using transposon sequencing. We demonstrate that in general the SOS response, several tRNA modifying enzymes as well as adaptation to microaerophilic growth conditions play a key role in bacterial survival under biofilm growth conditions. On the other hand, presence of genes involved in motility and PQS signaling were less important during biofilm growth. Several mutants exhibiting transposon insertions in genes detected in our screen were validated for their biofilm growth capabilities and biofilm specific transcriptional responses using independently generated transposon mutants. Our results provide new insights into P. aeruginosa adaptation to biofilm growth conditions. The detection of previously unknown determinants of biofilm survival supports the use of transposon insertion sequencing as a global genomic technology for understanding the establishment of difficult to treat biofilm-associated infections.

Exploring the complexity of host-pathogen communication is vital to understand why microbes persist within a host, while others are cleared. Here, we employed a dual-sequencing approach to unravel conversational turn-taking of dynamic host-pathogen communications. We demonstrate that upon hitting a host cell, motile Pseudomonas aeruginosa induce a specific gene expression program. This results in the expression of spermidine on the surface, which specifically activates the PIP3-pathway to induce phagocytic uptake into primary or immortalized murine cells. Non-motile bacteria are more immunogenic due to a lower expression of arnT upon host-cell contact, but do not produce spermidine and are phagocytosed less. We demonstrate that not only the presence of pathogen inherent molecular patterns induces immune responses, but that bacterial motility is linked to a host-cell-induced expression of additional immune modulators. Our results emphasize on the value of integrating microbiological and immunological findings to unravel complex and dynamic host-pathogen interactions.

Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Because different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here, we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single-nucleotide polymorphisms that are the basis for branching of the phylogenetic tree. We also identified single-nucleotide polymorphisms that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. Although our results demonstrate that the Pseudomonas aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.