Yeast zinc cluster transcription factors involved in the switch from fermentation to respiration show interdependency for DNA binding revealing a novel type of DNA recognition.

In budding yeast, fermentation is the most important pathway for energy production. Under low-glucose conditions, ethanol is used for synthesis of this sugar requiring a shift to respiration. This process is controlled by the transcriptional regulators Cat8, Sip4, Rds2 and Ert1. We characterized Gsm1 (glucose starvation modulator 1), a paralog of Rds2 and Ert1. Genome-wide analysis showed that Gsm1 has a DNA binding profile highly similar to Rds2. Binding of Gsm1 and Rds2 is interdependent at the gluconeogenic gene FBP1. However, Rds2 is required for Gsm1 to bind at other promoters but not the reverse. Gsm1 and Rds2 also bind to DNA independently of each other. Western blot analysis revealed that Rds2 controls expression of Gsm1. In addition, we showed that the DNA binding domains of Gsm1 and Rds2 bind cooperatively in vitro to the FBP1 promoter. In contrast, at the HAP4 gene, Ert1 cooperates with Rds2 for DNA binding. Mutational analysis suggests that Gsm1/Rds2 and Ert1/Rds2 bind to short common DNA stretches, revealing a novel mode of binding for this class of factors. Two-point mutations in a HAP4 site convert it to a Gsm1 binding site. Thus, Rds2 controls binding of Gsm1 at many promoters by two different mechanisms: regulation of Gsm1 levels and increased DNA binding by formation of heterodimers.

Antibiotic resistance gene sequencing is necessary to reveal the complex dynamics of immigration from sewers to activated sludge.

Microbial community composition has increasingly emerged as a key determinant of antibiotic resistance gene (ARG) content. However, in activated sludge wastewater treatment plants (AS-WWTPs), a comprehensive understanding of the microbial community assembly process and its impact on the persistence of antimicrobial resistance (AMR) remains elusive. An important part of this process is the immigration dynamics (or community coalescence) between the influent and activated sludge. While the influent wastewater contains a plethora of ARGs, the persistence of a given ARG depends initially on the immigration success of the carrying population, and the possible horizontal transfer to indigenously resident populations of the WWTP. The current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the influent microbial populations to reveal the fundamental mechanisms involved in ARG immigration between sewers and AS-WWTP. A novel multiplexed amplicon sequencing approach was used to track different ARG sequence variants across the immigration interface, and droplet digital PCR was used to quantify the impact of immigration on the abundance of the targeted ARGs. Immigration caused an increase in the abundance of over 70 % of the quantified ARGs. However, monitoring of ARG amplicon sequence variants (ARG-ASVs) at the immigration interface revealed various immigration patterns such as (i) suppression of the indigenous mixed liquor ARG-ASV by the immigrant, or conversely (ii) complete immigration failure of the influent ARG-ASV. These immigration profiles are reported for the first time here and highlight the crucial information that can be gained using our novel multiplex amplicon sequencing techniques. Future studies aiming to reduce AMR in WWTPs should consider the impact of influent immigration in process optimisation and design.

A lacZ reporter with high activity in the human fungal pathogen Candida albicans.

Reporter genes are useful tools to study gene transcription in various organisms. For example, the lacZ gene encoding ß-galactosidase has been extensively used as a reporter in bacteria, budding yeast, fruit fly, mouse etc. However, use of this gene in the human fungal pathogen Candida albicans has been limited, probably due to low ß-galactosidase activity. Here, we describe a reporter derived from the Vibrio cholerae lacZ gene in which codons have been optimized for expression in C. albicans. The constitutively active ACT1 promoter was fused to this synthetic lacZ reporter and integrated in the C. albicans genome. High ß-galactosidase activity in liquid assays was observed for this reporter as well as coloration on X-gal plates. When the lacZ reporter expression was driven by the MET3 promoter, ß-galactosidase activity in liquid assays and coloration on X-gal plates was higher in the absence of methionine, thus recapitulating the regulation of the native MET3 gene. This synthetic lacZ gene extends the toolbox of C. albicans reagents by providing a useful reporter for analysis of promoter activity in this organism of medical importance.

Genes associated with cognitive performance in the Morris water maze: an RNA-seq study.

Learning and memory are among higher-order cognitive functions that are based on numerous molecular processes including changes in the expression of genes. To identify genes associated with learning and memory formation, here, we used the RNA-seq (high-throughput mRNA sequencing) technology to compare hippocampal transcriptomes between mice with high and low Morris water maze (MWM) cognitive performance. We identified 88 differentially expressed genes (DEGs) and 24 differentially alternatively spliced transcripts between the high- and low-MWM-performance mice. Although the sets of DEGs and differentially alternatively spliced transcripts did not overlap, both were found to be enriched with genes related to the same type of biological processes: trans-synaptic signaling, cognition, and glutamatergic transmission. These findings were supported by the results of weighted-gene co-expression network analysis (WGCNA) revealing the enrichment of MWM-cognitive-performance-correlating gene modules with very similar Gene Ontology terms. High-MWM-performance mice manifested mostly higher expression of the genes associated with glutamatergic transmission and long-term potentiation implementation, which are processes necessary for memory acquisition and consolidation. In this set, there were genes participating in the regulation of trans-synaptic signaling, primarily AMPA receptor signaling (Nrn1, Nptx1, Homer3, Prkce, Napa, Camk2b, Syt7, and Nrgn) and calcium turnover (Hpca, Caln1, Orai2, Cpne4, and Cpne9). In high-MWM-performance mice, we also demonstrated significant upregulation of the "flip" splice variant of Gria1 and Gria2 transcripts encoding subunits of AMPA receptor. Altogether, our data helped to identify specific genes in the hippocampus that are associated with learning and long-term memory. We hypothesized that the differences in MWM cognitive performance between the mouse groups are linked with increased long-term potentiation, which is mainly mediated by increased glutamatergic transmission, primarily AMPA receptor signaling.

Repeated lipopolysaccharide exposure leads to placental endotoxin tolerance.

PROBLEM: Placental infection induces increased levels of pro-inflammatory cytokines, which have been implicated in the pathogenesis of pre-term labor. Endotoxin tolerance is a phenomenon in which exposure to a dose of endotoxin makes tissue less responsive to subsequent exposures. The objective of our study was to determine whether repeated exposure to endotoxin will induce a tolerant phenotype in normal human second-trimester placental tissue. METHODS OF STUDY: Human second-trimester placental explants from elective termination of pregnancy were cultured and exposed to endotoxin (LPS). After 24 hours, the media was collected for analysis, and the explants were re-exposed to LPS after adding fresh media for another 24 hours. This process was repeated for a total of 4 LPS doses. The media was collected from each day and analyzed for cytokine levels. RESULTS: The first LPS treatment stimulated the secretion of the pro-inflammatory cytokines IL-1ß and TNF-α. However, their production was significantly diminished with repeated LPS doses. Production of the anti-inflammatory cytokines, IL-1ra and IL-10, was also stimulated by the first LPS treatment, but secretion was more gradually and moderately decreased with repeated LPS doses compared to the pro-inflammatory cytokines. The ratios of the anti-inflammatory/pro-inflammatory mediators (IL-1ra/IL-1ß and IL-10/TNF-α) indicate a progressively more anti-inflammatory milieu with repeated LPS doses. CONCLUSION: Repeated LPS exposure of human second-trimester placental tissues induced endotoxin tolerance. We speculate that endotoxin tolerance at the maternal-fetal interface will protect the fetus from exaggerated inflammatory responses after repeated infectious exposure.

The switch from fermentation to respiration in Saccharomyces cerevisiae is regulated by the Ert1 transcriptional activator/repressor.

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene.

Phenotypic analysis of a family of transcriptional regulators, the zinc cluster proteins, in the human fungal pathogen Candida glabrata.

Candida glabrata is the second most important human fungal pathogen. Despite its formal name, C. glabrata is in fact more closely related to the nonpathogenic budding yeast Saccharomyces cerevisiae. However, less is known about the biology of this pathogen. Zinc cluster proteins form a large family of transcriptional regulators involved in the regulation of numerous processes such as the control of the metabolism of sugars, amino acids, fatty acids, as well as drug resistance. The C. glabrata genome encodes 41 known or putative zinc cluster proteins, and the majority of them are uncharacterized. We have generated a panel of strains carrying individual deletions of zinc cluster genes. Using a novel approach relying on tetracycline for conditional expression in C. glabrata at the translational level, we show that only two zinc cluster genes are essential. We have performed phenotypic analysis of nonessential zinc cluster genes. Our results show that two deletion strains are thermosensitive whereas two strains are sensitive to caffeine, an inhibitor of the target of rapamycin pathway. Increased salt tolerance has been observed for eight deletion strains, whereas one strain showed reduced tolerance to salt. We have also identified a number of strains with increased susceptibility to the antifungal drugs fluconazole and ketoconazole. Interestingly, one deletion strain showed decreased susceptibility to the antifungal micafungin. In summary, we have assigned phenotypes to more than half of the zinc cluster genes in C. glabrata. Our study provides a resource that will be useful to better understand the biological role of these transcription factors.

Does carbon monoxide inhibit proinflammatory cytokine production by fetal membranes?

AIM: Infection-induced inflammation is a common cause of preterm birth. Pharmacologic inhibition of proinflammatory cytokines improves pregnancy outcome in animal models but there are no universally effective therapies for preterm birth in women. Carbon monoxide (CO) has anti-inflammatory properties at low concentrations but its effects on reproductive tissues is unclear. Therefore, we studied the effect of supplemental CO on the production of cytokines associated with preterm birth by fetal membranes. METHODS: Cross-sections of whole fetal membranes, isolated choriodecidua, and isolated amnion were prepared using tissues collected from women who had normal vaginal deliveries at term. Tissues were placed in an organ explant culture system and stimulated with up to 10(8) CFU/mL Escherichia coli. Cultures were incubated under room air or room air+250 ppm CO for 18 h and cytokine concentrations in conditioned medium were quantified by ELISA. RESULTS: CO inhibited IL-1ß and TNF-α (P≤0.001) production by cultures stimulated with 10(7) CFU/mL bacteria but had no detectable effect on IL-10 by full-thickness membranes. Although CO also tended to reduce TNF-α production (P=0.053), no effect of CO was detected for IL-10 or IL-1ß for membranes stimulated with 10(8) CFU/mL E. coli. TNF-α, but not IL-1ß or IL-10 production, was inhibited by CO for choriodecidual cultures stimulated with 10(7) or 10(8) CFU/mL E. coli (P<0.001). IL-1ß production was significantly inhibited by CO for amnion cultures stimulated with 10(7) (P=0.002) and 10(8) (P=0.017) CFU/mL E. coli. Exposure to bacteria had no effect on TNF-α or IL-10 production but CO tended to increase IL-10 production by amnion cultures stimulated with 10(8) CFU/mL E. coli (P=0.037). CONCLUSIONS: These results suggest that CO may help promote an anti-inflammatory environment during intrauterine infections by inhibiting TNF-α and IL-1ß production.

Effect of oxygen tension on bacteria-stimulated cytokine production by fetal membranes.

AIM: Tissue culture studies indicate that bacterial products stimulate the production of proinflammatory cytokines by reproductive tissues. However, most of these studies have been performed under room air conditions, supplemented with 5% CO2. In this study, we tested whether O2 tension affects bacteria-stimulated cytokine production by extra-placental fetal membranes. METHODS: Cultures of full-thickness membranes, isolated choriodecidua, and isolated amnion were exposed to bacteria and incubated under 21% (room air) or 5% O2 for 18 h. Cytokine concentrations in conditioned medium was quantified by immunoassay. RESULTS: Culture under 5% O2 increased production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, but reduced IL-10 and IL-6 production by full membranes. Isolated choriodecidua responded to 5% O2 with increased IL-1ß production and reduced IL-6 production, but had no effect on TNF-α and IL-10 production was not detected. No effect of O2 tension on IL-1ß or IL-6 production by isolated amnion was detected, however, Escherichia coli-stimulated IL-10, TNF-α and IL-8 production was enhanced by culture under 5% O2. CONCLUSIONS: Increased oxygen tension reduces the pro-inflammatory responsiveness of cell cultures to E. coli and promotes an anti-inflammatory cytokine profile. Differential effects of O2 tension on choriodecidua and amnion suggests a network of paracrine factors that regulate cytokine levels in response to changes in O2 tension.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances placental inflammation.

Preterm birth is a leading cause of perinatal morbidity and mortality that is often associated with ascending infections from the lower genital tract. Recent studies with animal models have suggested that developmental exposure to the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can increase the risk of preterm birth in the offspring. How TCDD may modify placental immunity to ascending infections is unclear. Therefore, we studied the effects of TCDD treatment on basal and Escherichia coli-stimulated cytokine production by placental explants. Cultures of second-trimester placentas were treated with up to 40 nM TCDD for 72 h and then stimulated with 10(7)CFU/ml E. coli for an additional 24h. Concentrations of cytokines and PGE2 were measured in conditioned medium by immunoassay. TCDD exposure increased mRNA levels of IL-1ß by unstimulated cultures, but no effects on protein levels of this cytokine were detected. TNF-α production was unaffected by TCDD for unstimulated cultures, but pre-treatment with 40 nM TCDD significantly increased E. coli-stimulated TNF-α production. Both basal and bacteria-stimulated PGE2 and COX-2 gene expression were enhanced by TCDD pretreatment. In contrast, production of the anti-inflammatory cytokine, IL-10, was reduced by TCDD pretreatment for both unstimulated and E. coli-stimulated cultures. No effect of TCDD on the viability of the cultures was detected. These results suggest that TCDD exposure may shift immunity to enhance a proinflammatory phenotype at the maternal-fetal interface that could increase the risk of infection-mediated preterm birth.

Can carbon monoxide prevent infection-mediated preterm birth in a mouse model?

PROBLEM: Preterm birth is frequently caused by intrauterine infection and inflammation. Recent studies have demonstrated that carbon monoxide (CO), which is produced endogenously, has potent anti-inflammatory properties. Whether or not CO can prevent infection-mediated preterm birth is unknown. METHODS: Mice were assigned to one of four groups: sham infection, sham infection + CO, infection, or infection + CO. Infections were established by intra-uterine injection of Escherichia coli on day 14 of pregnancy. Animals received daily i.p. injections of 1 mL CO-saturated lactated ringers solution (LRS) or LRS alone beginning on the morning of surgery. Gestational age at delivery and litter characteristics was noted. In second experiment, animals were sacrificed 24 hrs post-surgery and tissues were harvested for cytokine analyses. RESULTS: Escherichia coli intrauterine infection increased the number of animals delivering preterm. This effect was significantly ameliorated by CO-LRS. CO-treatment also increased litter size and weights of the surviving offspring. Cytokines in the amniotic fluid and the placenta were increased by E. coli exposure, but CO had no detectible effect on E. coli-stimulated cytokine production. No effects of CO were detected in sham-infected animals. CONCLUSION: Supplemental CO improves pregnancy outcome after intrauterine infection and may function at a point downstream of, or through pathways independent of, induction of proinflammatory cytokines.

Effect of carbon monoxide on bacteria-stimulated cytokine production by placental explants.

PROBLEM: Preterm birth is frequently caused by an inflammatory response to ascending infections of the reproductive tract. Carbon monoxide (CO) has potent anti-inflammatory properties at subtoxic concentrations. Whether or not CO can modulate inflammatory responses by placental tissues is unclear. METHODS: Placental explant cultures were incubated with heat-killed Escherichia coli or Ureaplasma parvum in the presence or absence of 250 ppm CO for 24 hr. Concentrations of cytokines relative viability of the cultures were quantified. RESULTS: Escherichia coli- and U. parvum-stimulated IL-1ß production was significantly inhibited by CO supplementation. Escherichia coli-stimulated, but not U. parvum-stimulated, IFN-γ production was inhibited by CO. While CO inhibited PGE(2) production by unstimulated cells, no effects on bacteria-stimulated prostaglandin production were detected. CO had no effect on basal or E. coli-stimulated TNF-α production but enhanced TNF-α production by cultures stimulated with U. parvum. In addition, CO tended to improve the viability of the placental cultures. CONCLUSIONS: Low concentrations of CO tended to reduce proinflammatory cytokines and to promote the production of anti-inflammatory cytokines in a pathogen-specific manner. These properties suggest that CO may be useful for promoting a pro-pregnancy cytokine milieu by placental explants and may reduce the consequences of intrauterine infections.