A molecular mechanism for the "digital" response of p53 to stress.

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

The complex architecture of p53 binding sites.

Sequence-specific protein-DNA interactions are at the heart of the response of the tumor-suppressor p53 to numerous physiological and stress-related signals. Large variability has been previously reported in p53 binding to and transactivating from p53 response elements (REs) due, at least in part, to changes in direct (base) and indirect (shape) readouts of p53 REs. Here, we dissect p53 REs to decipher the mechanism by which p53 optimizes this highly regulated variable level of interaction with its DNA binding sites. We show that hemi-specific binding is more prevalent in p53 REs than previously envisioned. We reveal that sequences flanking the REs modulate p53 binding and activity and show that these effects extend to 4-5 bp from the REs. Moreover, we show here that the arrangement of p53 half-sites within its REs, relative to transcription direction, has been fine-tuned by selection pressure to optimize and regulate the response levels from p53 REs. This directionality in the REs arrangement is at least partly encoded in the structural properties of the REs. Furthermore, we show here that in the p21-5' RE the orientation of the half-sites is such that the effect of the flanking sequences is minimized and we discuss its advantages.

From Survival to Productivity Mode: Cytokinins Allow Avoiding the Avoidance Strategy Under Stress Conditions.

Growth retardation and stress-induced premature plant senescence are accompanied by a severe yield reduction and raise a major agro-economic concern. To improve biomass and yield in agricultural crops under mild stress conditions, the survival must be changed to productivity mode. Our previous successful attempts to delay premature senescence and growth inhibition under abiotic stress conditions by autoregulation of cytokinins (CKs) levels constitute a generic technology toward the development of highly productive plants. Since this technology is based on the induction of CKs synthesis during the age-dependent senescence phase by a senescence-specific promoter (SARK), which is not necessarily regulated by abiotic stress conditions, we developed autoregulating transgenic plants expressing the IPT gene specifically under abiotic stress conditions. The Arabidopsis promoter of the stress-induced metallothionein gene (AtMT) was isolated, fused to the IPT gene and transformed into tobacco plants. The MT:IPT transgenic tobacco plants displayed comparable elevated biomass productivity and maintained growth under drought conditions. To decipher the role and the molecular mechanisms of CKs in reverting the survival transcriptional program to a sustainable plant growth program, we performed gene expression analysis of candidate stress-related genes and found unexpectedly clear downregulation in the CK-overproducing plants. We also investigated kinase activity after applying exogenous CKs to tobacco cell suspensions that were grown in salinity stress. In-gel kinase activity analysis demonstrated CK-dependent deactivation of several stress-related kinases including two of the MAPK components, SIPK and WIPK and the NtOSAK, a member of SnRK2 kinase family, a key component of the ABA signaling cascade. A comprehensive phosphoproteomics analysis of tobacco cells, treated with exogenous CKs under salinity-stress conditions indicated that >50% of the identified phosphoproteins involved in stress responses were dephosphorylated by CKs. We hypothesize that upregulation of CK levels under stress conditions desensitize stress signaling cues through deactivation of kinases that are normally activated under stress conditions. CK-dependent desensitization of environmental stimuli is suggested to attenuate various pathways of the avoidance syndrome including the characteristic growth arrest and the premature senescence while allowing normal growth and metabolic maintenance.

Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

[This corrects the article DOI: 10.3389/fmicb.2017.02613.].

Murine Genetic Background Has a Stronger Impact on the Composition of the Gut Microbiota than Maternal Inoculation or Exposure to Unlike Exogenous Microbiota.

The gut microbiota is a complex ecosystem, affected by both environmental factors and host genetics. Here, we aim at uncovering the bacterial taxa whose gut persistence is controlled by host genetic variation. We used a murine model based on inbred lines BALB/c and C57BL/6J and their F1 reciprocal hybrids (♀C57BL/6J × â™‚BALB/c; ♀BALB/c × â™‚C57BL/6J). To guarantee genetic similarity of F1 offspring, including the sex chromosomes, we used only female mice. Based on 16S rRNA gene sequencing, we found that the genetically different inbred lines present different microbiota, whereas their genetically identical F1 reciprocal hybrids presented similar microbiota. Moreover, the F1 microbial composition differed from that of both parental lines. Twelve taxa were shown to have genetically controlled gut persistence, while none were found to show maternal effects. Nine of these taxa were dominantly inherited by the C57BL/6J line. Cohousing of the parental inbred lines resulted in a temporary and minor shift in microbiota composition, which returned back to the former microbial composition following separation, indicating that each line tends to maintain a unique bacterial signature reflecting the line. Taken together, our findings indicate that mouse genetics has an effect on the microbial composition in the gut, which is greater than maternal effect and continuous exposure to different microbiota of the alternative line. Uncovering the bacterial taxa associated with host genetics and understanding their role in the gut ecosystem could lead to the development of genetically oriented probiotic products, as part of the personalized medicine approach.IMPORTANCE The gut microbiota play important roles for their host. The link between host genetics and their microbial composition has received increasing interest. Using a unique reciprocal cross model, generating genetically similar F1 hybrids with different maternal inoculation, we demonstrate the inheritance of gut persistence of 12 bacterial taxa. No taxa identified as maternally transmitted. Moreover, cohabitation of two genetically different inbred lines did not dramatically affect the microbiota composition. Taken together, our results demonstrate the importance of the genetic effect over maternal inoculation or effect of exposure to unlike exogenous microbiota. These findings may lead to the development of personalized probiotic products, specifically designed according to the genetic makeup.

Radiation induces proinflammatory dysbiosis: transmission of inflammatory susceptibility by host cytokine induction.

OBJECTIVE: Radiation proctitis (RP) is a complication of pelvic radiotherapy which affects both the host and microbiota. Herein we assessed the radiation effect on microbiota and its relationship to tissue damage using a rectal radiation mouse model. DESIGN: We evaluated luminal and mucosa-associated dysbiosis in irradiated and control mice at two postradiation time points and correlated it with clinical and immunological parameters. Epithelial cytokine response was evaluated using bacterial-epithelial co-cultures. Subsequently, germ-free (GF) mice were colonised with postradiation microbiota and controls and exposed to radiation, or dextran sulfate-sodium (DSS). Interleukin (IL)-1ß correlated with tissue damage and was induced by dysbiosis. Therefore, we tested its direct role in radiation-induced damage by IL-1 receptor antagonist administration to irradiated mice. RESULTS: A postradiation shift in microbiota was observed. A unique microbial signature correlated with histopathology. Increased colonic tumor necrosis factor (TNF)α, IL-1ß and IL-6 expression was observed at two different time points. Adherent microbiota from RP differed from those in uninvolved segments and was associated with tissue damage. Using bacterial-epithelial co-cultures, postradiation microbiota enhanced IL-1ß and TNFα expression compared with naïve microbiota. GF mice colonisation by irradiated microbiota versus controls predisposed mice to both radiation injury and DSS-induced colitis. IL-1 receptor antagonist administration ameliorated intestinal radiation injury. CONCLUSIONS: The results demonstrate that rectal radiation induces dysbiosis, which transmits radiation and inflammatory susceptibility and provide evidence that microbial-induced radiation tissue damage is at least in part mediated by IL-1ß. Environmental factors may affect the host via modifications of the microbiome and potentially allow for novel interventional approaches via its manipulation.

Draft Genome Sequence of Salmonella enterica Serovar Senftenberg 070885 and Its Linalool-Adapted Mutant.

Here we report the genome sequences of both Salmonella Senftenberg 070885, a clinical isolate from the 2007 outbreak linked to basil, and its mutant linalool-adapted S Senftenberg (LASS). These draft genomes of S Senftenberg may enable the identification of bacterial genes responsible for resistance to basil oil.

Phylogeny of Vibrio vulnificus from the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name "piscis."

Acrolein increases macrophage atherogenicity in association with gut microbiota remodeling in atherosclerotic mice: protective role for the polyphenol-rich pomegranate juice.

The unsaturated aldehyde acrolein is pro-atherogenic, and the polyphenol-rich pomegranate juice (PJ), known for its anti-oxidative/anti-atherogenic properties, inhibits macrophage foam cell formation, the hallmark feature of early atherosclerosis. This study aimed to investigate two unexplored areas of acrolein atherogenicity: macrophage lipid metabolism and the gut microbiota composition. The protective effects of PJ against acrolein atherogenicity were also evaluated. Atherosclerotic apolipoprotein E-deficient (apoE-/-) mice that were fed acrolein (3 mg/kg/day) for 1 month showed significant increases in serum and aortic cholesterol, triglycerides, and lipid peroxides. In peritoneal macrophages isolated from the mice and in J774A.1 cultured macrophages, acrolein exposure increased intracellular oxidative stress and stimulated cholesterol and triglyceride accumulation via enhanced rates of their biosynthesis and over-expression of key regulators of cellular lipid biosynthesis: sterol regulatory element-binding proteins (SREBPs), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and diacylglycerol acyltransferase1 (DGAT1). Acrolein-fed mice demonstrated a major shift in the gut microbiota composition, including a significant phylum-level change in increased Firmicutes and decreased Bacteroidetes. At the family level, acrolein significantly increased the prevalence of Ruminococcaceae and Lachnospiraceae of which the Coprococcus genus was significantly and positively correlated with serum, aortic and macrophage lipid levels and peroxidation. The pro-atherogenic effects of acrolein on serum, aortas, macrophages, and the gut microbiota were substantially abolished by PJ. In conclusion, these findings provide novel mechanisms by which acrolein increases macrophage lipid accumulation and alters the gut microbiota composition in association with enhanced atherogenesis. Moreover, PJ was found as an effective strategy against acrolein atherogenicity.

Draft Genome Sequence of Lactobacillus johnsonii Strain 16, Isolated from Mice.

Here, we report the genome sequence of Lactobacillus johnsonii, a member of the gut lactobacilli. This draft genome of L. johnsonii strain 16 isolated from C57BL/6J mice enables the identification of bacterial genes responsible for host-specific gut persistence.

Draft Genome Sequence of the Pathogenic Bacterium Vibrio vulnificus V252 Biotype 1, Isolated in Israel.

We report the genome sequence of the pathogenic Vibrio vulnificus biotype 1 clade B, which is suggested to have a common ancestor with biotype 3. This draft genome of the clinical strain V252, isolated in Israel, represents the clonal clade B group that contains both clinical and environmental strains.

Draft Genome Sequence of Environmental Bacterium Vibrio vulnificus CladeA-yb158.

We report the genome sequence of the environmental Vibrio vulnificus biotype 1_cladeA. This draft genome of the CladeA-yb158 strain, isolated in Israel, represents this newly emerged clonal group that contains both clinical and environmental strains.

Genome-wide SNP-genotyping array to study the evolution of the human pathogen Vibrio vulnificus biotype 3.

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen.

Biodiversity of Enterococcus faecalis based on genomic typing.

Enterococcus faecalis is a common inhabitant of the gastrointestinal tracts of different animals and is also found in other environments, such as plants, soil, food and water. The diverse nature of E. faecalis, which includes pathogenic, commensal and probiotic strains, calls for the development of tools for accurate discrimination and characterization at the strain level. Here we studied the genetic relationships among 106 E. faecalis strains isolated from diverse origins and possessing different degrees of virulence. Strain typing was conducted using a set of selected simple-sequence repeat (SSR) loci combined with multilocus sequence typing (MLST) analysis, which discriminated among the strains and separated them into three main clusters. While pathogenic and commensal isolates were dispersed along the dendrogram, probiotic and cheese-originated strains were highly associated with one specific cluster (cluster 1). The strain panel was further characterized by testing the occurrence of two virulence determinants (esp gene and ß-hemolysis). The two determinants showed low abundance among probiotic and cheese-originated strains within cluster 1 when compared to non-cluster 1 cheese-originated strains, indicating a possible association of cluster 1 with non-virulent strains. Our results further emphasize the importance and challenge of precise characterization of E. faecalis strains from various sources.

Genome Sequence of the Pathogenic Bacterium Vibrio vulnificus Biotype 3.

We report the first genome sequence of the pathogenic Vibrio vulnificus biotype 3. This draft genome sequence of the environmental strain VVyb1(BT3), isolated in Israel, provides a representation of this newly emerged clonal group, which reveals higher similarity to the clinical strains of biotype 1 than to the environmental ones.

Insight into the evolution of Vibrio vulnificus biotype 3's genome.

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are biochemically classified into three biotypes. The newly emerged biotype 3 appears to be rather clonal and geographically restricted to Israel, where it caused an outbreak of wound infections and bacteremia. To understand the evolution of the bacterium's genome, we sequenced and analyzed the genome of biotype 3 strain VVyb1(BT3), and then conducted a microbial environmental survey of the hypothesized niche from which it probably evolved. The genome of this environmental isolate revealed higher similarity to the published biotype 1 genomes of clinical strains (90%) than to the environmental strains (87%), supporting the virulence of the biotype 3 group. Moreover, 214 of the total 5361 genes were found to be unique to strain VVyb1(BT3), having no sequence similarity to any of the known genomes of V. vulnificus; 35 of them function in DNA mobility and rearrangement, supporting the role of horizontal gene transfer in genome evolution. Interestingly, 29 of the "unique" genes had homologies among Shewanella species. In a survey conducted in aquaculture ponds in Israel, we successfully co-isolated Shewanella and V. vulnificus from the same niche, further supporting the probable contribution of Shewanella to the genome evolution of biotype 3. Indeed, one gene was found in a S. algae isolate. Surprisingly, molecular analysis revealed that some of the considered unique genes are harbored by non-sequenced biotype 1 strains isolated from the same environment. Finally, analyses of the biotype 3 genome together with the environmental survey suggested that its genome originated from a biotype 1 Israeli strain that acquired a rather small number of genes from other bacterial species in the niche, such as Shewanella. Therefore, aquaculture is likely to play a major role as a man-made ecological niche in bacterial evolution, leading the emergence of new pathogenic groups in V. vulnificus.

Indication for Co-evolution of Lactobacillus johnsonii with its hosts.

BACKGROUND: The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. RESULTS: A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice. CONCLUSIONS: Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.

Genetic modification of iron metabolism in mice affects the gut microbiota.

The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe-/-, Irp2-/- and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2-/- mice compared to wild type and Hfe-/- mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2-/- and Hfe-/- mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L. intestinalis were highly abundant in Irp2-/- mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.

Genetic diversity of the human pathogen Vibrio vulnificus: a new phylogroup.

The biotype 3 group of the human pathogen Vibrio vulnificus emerged in Israel probably as a result of genome hybridization of two bacterial populations. We performed a genomic and phylogenetic study of V. vulnificus strains isolated from the environmental niche from which this group emerged - fish aquaculture in Israel. The genetic relationships and evolutionary aspects of 188 environmental and clinical isolates of the bacterium were studied by genomic typing. Genetic relations were determined based on variation at 12 variable number tandem repeat (VNTR, also termed SSR) loci. Analysis revealed a new cluster, in addition to the main groups of biotype 1& 2 and biotype 3. Similar grouping results were obtained with three different statistical approaches. Isolates forming this new cluster presented unclear biochemical profile nevertheless were not identified as biotype 1 or biotype 3. Further examination of representative strains by multilocus sequence typing (MLST) of 10 housekeeping genes and 5 conserved hypothetical genes supported the identification of this as yet undiscovered phylogroup (phenotypically diverse), termed clade A herein. This new clonal subgroup includes environmental as well as clinical isolates. The results highlight the fish aquaculture environment, and possibly man-made ecological niches as a whole, as a source for the emergence of new pathogenic strains.

Vibrio cholerae autoinducer CAI-1 interferes with Pseudomonas aeruginosa quorum sensing and inhibits its growth.

The human pathogen Vibrio cholerae uses several small molecules to coordinate gene expression in a process termed quorum sensing (QS), and its main autoinducer is CAI-1. We have examined the activity of this signaling molecule in three other species of bacteria. Interestingly, while showing an inhibitory effect on QS in the opportunistic pathogen P. aeruginosa at low micromolar concentrations, it caused also growth inhibition at higher concentrations. In contrast, the two other bacteria were unaffected, and we suggest a possible mechanism for these effects, based on membrane perturbation studies.