A genome-wide cytotoxicity screen of cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth.

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

Isolation and Annotation of Azira, a CT Cluster Phage That Infects Gordonia rubripertincta.

Azira is a CT cluster actinobacteriophage that infects Gordonia rubripertincta NRRL B-16540. The genome contains 67 predicted protein coding genes, of which 31 have a putative function. Azira has a lysis cassette encoding two endolysins and three transmembrane proteins. Azira contains four genes predicted to encode enzymes involved in thymine synthesis.

Annotation of Secretariat and Hydrus, two DJ cluster phages isolated on Gordonia rubripertincta.

Secretariat and Hydrus are phages grouped into the DJ cluster that were isolated on Gordonia rubripertincta NRRL B-16540. The phages have 75% nucleotide identity and share 73% gene content. Secretariat has a genome with 84 predicted genes, while Hydrus has 91 predicted genes and can also infect Gordonia terrae 3612.

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands.

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta.

Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some models, generate large "pores" in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive Gordonia rubripertincta NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1-4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered.

Genome Sequence of CaiB, a DR Cluster Actinobacteriophage That Infects Gordonia rubripertincta.

CaiB is a DR cluster actinobacteriophage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 61,620 bp, has a GC content of 68.6%, and contains 85 predicted protein coding genes. CaiB has several putative operons and has repeated intergenic regions that may be involved in gene regulation.

Genome Sequence of VanLee, a Singleton Actinobacteriophage That Infects Multiple Gordonia Strains.

VanLee is a singleton phage that was isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540 as the host. The genome is 84,560 bp and has a GC content of 67.8%. VanLee has 164 predicted protein-coding genes and one tRNA. VanLee can infect Gordonia terrae with the same efficiency as G. rubripertincta.

Genome Sequences of Akoni, Ashton, and Truong, Podoviridae Bacteriophages Isolated from Microbacterium foliorum.

Cluster EK2 Akoni, Ashton, and Truong are lytic Podoviridae actinobacteriophages that were isolated from soil in Florida using Microbacterium foliorum NRRL B-24224 as the host. The genomes are 54,307 bp, 54,560 bp, and 54,309 bp, respectively, and are 60% GC rich. Each genome contains a novel 13,464-bp gene that encompasses 25% of the genome.

Genomic diversity of bacteriophages infecting Microbacterium spp.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Evaluating Psychosocial Mechanisms Underlying STEM Persistence in Undergraduates: Scalability and Longitudinal Analysis of Three Cohorts from a Six-Day Pre-College Engagement STEM Academy Program.

In a previous report, we validated that a cohort of first-year undergraduates who participated in a weeklong pre-college engagement STEM Academy (SA) program were retained in science, technology, engineering, and mathematics (STEM) at a higher rate than a matched comparison group (MCG). In addition, SA students yielded increases in science identity and sense of belonging to STEM and to the university. Here, we report the ability to scale the size of the SA program to accommodate more students and replicate the previous findings with two additional cohorts. Longitudinal analysis of the 2015 and 2016 program cohorts demonstrate that both groups were retained to STEM disciplines and the university at higher rates than a MCG. To assess what underlying psychological mechanisms lead to increases in science identity and university belonging, we tested three exploratory models. These models indicate that positive changes in university and STEM belonging indirectly predict an increase in science identity. Further, positive changes in perceived family support indirectly predict increases in university belonging. Thus, through the evaluation of three different cohorts, we found robust evidence that the SA program increases sense of belonging and science identity, and these attitudinal changes promote undergraduate persistence in STEM.

Repression of aryl hydrocarbon receptor (AHR) signaling by AHR repressor: role of DNA binding and competition for AHR nuclear translocator.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving "transrepression" of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.

Specific blockage of ligand-induced degradation of the Ah receptor by proteasome but not calpain inhibitors in cell culture lines from different species.

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Functional analysis of cis-regulatory regions within the dioxin-inducible CYP1A promoter/enhancer region from zebrafish (Danio rerio).

In vitro mutagenesis was utilized to render the various xenobiotic response elements (XREs) within the zebrafish CYP1A promoter/enhancer region non-functional either independently or in combination. Reporter gene assays revealed that only XRE4, XRE7, and XRE8 contributed to maximal TCDD-mediated induction of luciferase and that the contribution of each XRE to maximal induction was not equal. XRE4 and XRE7 were capable of functioning independently, while XRE8 alone could not support TCDD-mediated induction but was required for the ability of XRE4 and XRE7 to support maximal induction. These results were observed in cell lines derived from human, mouse and zebrafish. Mutagenesis of 3' nucleotides flanking the non-functional XRE5, and functional XRE4 did not alter the function of these XREs in cell culture. In silico analyses revealed the presence of putative Sp1, AP2, CREB, and two HNF-3 transcription factor binding sites that were localized to common positions within the enhancer region of both the mouse and zebrafish CYP1A genes. In vitro mutagenesis of the binding sites showed that loss of the Sp1 or AP2 sites had minimal impact on TCDD-mediated gene induction while loss of the putative CREB site resulted in a modest decrease in basal and inducible activity and mutation of the HNF-3 reduced inducible activity by >90% of controls. Collectively, these findings suggest that the presence of XREs is not the sole determinant for regulation of aryl hydrocarbon receptor (AHR)-mediated gene and do not function in an additive manner.

Evaluating Psychosocial Mechanisms Underlying STEM Persistence in Undergraduates: Evidence of Impact from a Six-Day Pre-College Engagement STEM Academy Program.

The persistence of undergraduate students in science, technology, engineering, and mathematics (STEM) disciplines is a national issue based on STEM workforce projections. We implemented a weeklong pre-college engagement STEM Academy (SA) program aimed at addressing several areas related to STEM retention. We validated an instrument that was developed based on existing, validated measures and examined several psychosocial constructs related to STEM (science identity, self-efficacy, sense of belonging to the university and to STEM, career expectancies, and intention to leave STEM majors) before and after the program. We also compared students in the SA program with a matched comparison group of first-year students. Results show that SA students significantly increased in science identity and sense of belonging to STEM and to the university, all predictive of increased STEM retention and a primary aim of the program. Relative to the matched comparison group, SA students began their first semester with higher STEM self-efficacy, sense of belonging, and science identity, positive career expectancies, and lower intention to leave STEM. The SA cohort showed 98% first-year retention and 92% STEM major retention. The SA program serves as a model of a scalable, first-level, cocurricular engagement experience to enhance psychosocial factors that impact undergraduate persistence in STEM.

Ligand-dependent and -independent degradation of the human aryl hydrocarbon receptor (hAHR) in cell culture models.

Studies have shown that zebrafish and rodent aryl hydrocarbon receptors (AHRs) are degraded following ligand exposure and that reductions in AHR protein can impact growth and development in vivo. The current study was designed to evaluate the degradation of the AHR in seven human cell lines that were derived from various carcinomas or from normal tissue. Consistent with studies in other species, the results show that the human AHR (hAHR) is degraded in a ligand dependent manner following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin for up to 16h. However, the hAHRs expressed in the various cell lines show differences in the time course and magnitude of degradation. The ligand dependent degradation is completely blocked by treatment with the proteasome inhibitor, MG-132. Ligand-independent degradation of the hAHR following exposure to geldanamycin (GA) is also observed in the different cell lines, although the magnitude of hAHR degradation is also is variable. These findings are significant since they indicate that ligand-dependent and independent degradation of the AHR is a conserved aspect of this signal transduction cascade from fish to human. In addition, the study identifies several cell lines that may provide novel models to further assess the regulation of AHR-mediated signaling and degradation of the human AHR.

Role of the carboxy-terminal transactivation domain and active transcription in the ligand-induced and ligand-independent degradation of the mouse Ahb-1 receptor.

To assess the importance of transactivation domains (TAD), DNA binding and transcription on the degradation of the AH receptor (AHR), Hepa-1 cells were pre-treated with actinomycin D (AD) or cycloheximide (CHX) and exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). AD or CHX did not affect nuclear localization or DNA binding of the AHR but inhibited ligand-induced degradation. In contrast, AD or CHX did not inhibit geldanamycin (GA) induced degradation of the AHR. To assess the role of the COOH-terminal TAD in AHR degradation, stop codons were placed at nucleotide 1501 and 1921 of the Ah(b-1) AHR coding region to generate AHR(500) and AHR(640). Stable cell lines were generated and exposed to TCDD. Cells expressing AHR(500) did not induce CYP1A1 protein, but exhibited significant degradation of AHR(500). Cells expressing AHR(640) induced CYP1A1 protein to 50% of the level of cells expressing wild type AHR and exhibited significant degradation of AHR(640). Importantly, AD and CHX did not inhibit the TCDD-induced degradation of either AHR(500) and AHR(640) and these receptors showed a more rapid profile of ligand-induced degradation compared to cells expressing wild type AHR. TCDD exposure to Hepa-1 cells with reduced aryl hydrocarbon receptor nuclear translocator (ARNT), showed ligand-induced degradation of the AHR that was not blocked by AD. However, AD inhibited TCDD-induced degradation when ARNT expression was restored. These results show that multiple mechanisms exist for the ligand and GA-induced degradation of the AHR and suggest that ligand-induced degradation can switch between two mechanisms depending on the presence of a functional TAD and the binding to DNA.

Functional characterization of aryl hydrocarbon receptor (zfAHR2) localization and degradation in zebrafish (Danio rerio).

The basic-helix-loop-helix/PAS (bHLH/PAS) family of proteins is a group of transcription factors that regulate key pathways during normal development and in the response to stress. The aryl hydrocarbon receptor (AHR) is a member of this family. Recently, Danio rerio (zebrafish) has become an important model system in the study of the signal transduction pathway and complements the results seen in mammalian models. However, studies of the AHR protein have been limited by the lack of antibody reagents and thus, little is known concerning the localization and degradation of the zebrafish AHR (zfAHR). In this report, we describe the production and characterization of specific polyclonal antibodies to the zfAHR2 protein and the analysis of AHR-mediated signal transduction in the zebrafish liver cell line (ZFL). The results show that the zfAHR2 is degraded via the 26S proteasome following exposure of cells to beta-naphthoflavone (BNF). Interestingly, the time course is slower and the magnitude of zfAHR2 degradation is not as great as seen for the mammalian AHR. Studies also show that the zfAHR2 is rapidly degraded in a ligand-independent manner by exposure of cells to geldanamycin (GA) to levels consistent with mammalian AHR. Finally, immunohistochemical staining of the ZFL cells suggest that the unliganded AHR resides in both the cytoplasm and nucleus and undergoes active nucleocytoplasmic shuttling in the absence of ligand. These results suggest that there is conservation of function between fish and mammals with respect to ligand-dependent and -independent degradation of the AHR and that the zfAHR2 is degraded via the 26S proteasome.

Analysis of rainbow trout Ah receptor protein isoforms in cell culture reveals conservation of function in Ah receptor-mediated signal transduction.

Two distinct aryl hydrocarbon receptor (AHR) cDNAs have been isolated from rainbow trout. The encoded receptor protein products termed rtAHR2alpha and rtAHR2ss are 97% identical at the amino acid level but are reported to have distinct functions with regard to AHR-mediated gene regulation. To test this hypothesis, the two proteins were evaluated functionally both in vitro and in a Chinese hamster lung cell line, E36. To facilitate analysis, both rtAHR2 isoforms were tagged with the FLAG peptide and could be expressed and quantified in a rabbit reticulocyte lysate. However, both proteins failed to form functional complexes with mammalian or rainbow trout AHR nuclear translocator protein (ARNT) that could associate with xenobiotic response elements (XREs) in a ligand-dependent manner in vitro. In contrast, both proteins exhibited positive function on AHR-mediated signaling when expressed in the E36 cell line. Both rtAHR2 isoforms showed a cytoplasmic distribution in the unliganded state and could drive the expression of a reporter gene under control of the trout CYP1A3 promoter. Although both proteins induced reporter gene activity to the same magnitude, the EC(50) values of the two isoforms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) differed by an order of magnitude, with the rtAHR2ss isoform less responsive to TCDD. When the functions of the rtAHR2 isoforms were tested in the context of the dominant negative rtARNT(a) protein, TCDD-mediated induction of reporter gene activity was reduced as the level of rtARNT(a) protein increased. In summary, both rtAHR2 isoforms appear to exhibit positive function in AHR-mediated signaling, suggesting conservation of function.

Linked expression of Ah receptor, ARNT, CYP1A1, and CYP1B1 in rat mammary epithelia, in vitro, is each substantially elevated by specific extracellular matrix interactions that precede branching morphogenesis.

Cytochrome P4501B1 (CYP1B1), the major constitutively expressed CYP in the rat mammary gland, is induced by Ah-receptor (AhR) ligands, while CYP1A1 is predominantly expressed only after induction. These CYPs contribute to carcinogenic activation of polycyclic aromatic hydrocarbons (PAHs). AhR, ARNT, and CYP1B1 were only weakly expressed, even after 2,3,7,8-tetrachlorodibenzo-p-dioxin induction, when rat mammary epithelial cells (RMEC) were cultured on plastic. RMEC cultured on the extracellular matrix (ECM), Matrigel, or on a floating gel of collagen I demonstrated branching morphogenesis and substantially increased basal CYP1B1 and induced CYP1A1 expression, in parallel with large increases in AhR and ARNT expression. Branching was more pronounced in the Wistar Kyoto than in the Wistar Furth rat strain. Although EGF enhanced branching, neither strain nor growth factor treatment substantially impacted CYP expression. Increased AhR and ARNT expression is observed within 24 h of dispersal on Matrigel, substantially prior to branch formation. Culture on thin layers of collagen I, collagen IV, and laminin, respectively, failed to reproduce the branching morphogenesis or increases in AhR, ARNT, or CYP expression. However, adherent, gelled collagen I recapitulated the increased protein expression, without supporting branching. This increased protein expression was closely paralleled by enhanced expression of beta-catenin and E-cadherin, components of cell-cell adhesion complexes. A synthetic peptide that selectively antagonizes integrin-ECM interactions reduced branch formation, without diminishing AhR, ARNT, and CYP expression. These data demonstrate that early ECM surface adhesion interactions mediate AhR and ARNT expression, which enhances CYP expression, independent of branching morphogenesis.

The mechanism of AH receptor protein down-regulation (degradation) and its impact on AH receptor-mediated gene regulation.

The proteolytic degradation of transcription factors is an established mechanism of regulating signal transduction pathways. Recent reports have suggested that the aryl hydrocarbon receptor (AHR) protein is rapidly downregulated (degraded) following ligand binding. The downregulation of AHR has been observed in nine distinct cells culture lines derived from human and rodent tissues and has also been observed in rodent models following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The downregulation of AHR appears to be ubiquitin mediated and occurs via the 26S proteasome pathway following nuclear export of AHR. The consequence of blocking AHR degradation in cell culture appears to be an increase in both the magnitude and duration of gene regulation by the AHR.ARNT complex. Thus, the physiological role of AHR degradation may be to modulate AHR-mediated gene regulation. This review provides analysis of the studies that have focused on the degradation of AHR in vivo and in vitro and the hypothesis that the downregulation of AHR is critical in the attenuation of AHR-mediated gene regulation.