Manipulation of the fungal epigenome is hypothesized to be an effective method for accessing natural products from silent biosynthetic pathways. A library of epigenetic modifiers was tested using the fungus Aspergillus niger to determine the impact of small-molecule inhibitors on reversing the transcriptional suppression of biosynthetic genes involved in polyketide (PKS), non-ribosomal peptide (NRPS), and hybrid PKS-NRPS (HPN) production. Examination of expressed sequence tag libraries from A. niger demonstrated that >70% of its PKS-, NRPS-, and HPN-encoding gene clusters were transcriptionally suppressed under standard laboratory culture conditions. Using a chemical epigenetic methodology, we showed that treatment of A. niger with suberoylanilide hydroxamic acid and 5-azacytidine led to the transcriptional upregulation of many secondary-metabolite-encoding biosynthetic gene clusters. Chemical epigenetic modifiers exhibited positional biases for upregulating chromosomally distal gene clusters. In addition, a phylogenetic-based preference was noted in the upregulation of reducing clade I PKS gene clusters, while reducing clade IV PKS gene clusters were largely unaffected. Manipulating epigenetic features in fungi is a powerful method for accessing the products of silent biosynthetic pathways. Moreover, this approach can be readily incorporated into modern microbial screening operations.

The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H 2 O 2 ) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H 2 O 2 .

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies-temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)-to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.The acquisition of iron is essential for bacterial pathogenesis. Because iron is insoluble in aqueous environments at neutral pH and has the potential to produce damaging free radicals, the mammalian host tightly sequesters iron (1, 51), lowering the level of free iron available to invading microorganisms to well below what is needed to survive (34). Since survival in the host and hence virulence is dependent on the acquisition of iron, pathogenic bacteria must be able to sense iron availability and globally regulate the transcription of iron acquisition genes. For many gram-positive and gram-negative organisms, including the sexually transmitted disease pathogen Neisseria gonorrhoeae, the crucial balance between acquiring enough iron to grow and avoiding the toxic effects of excess iron is controlled by the ferric uptake regulator (Fur) protein (12, 27, 49). Typically, Fur acts as a transcriptional repressor by binding as a dimer, along with ferrous iron as a corepressor, to regulatory Fur box (FB) sequences in the promoters of iron-regulated genes under iron-rich growth conditions. As intracellular iron stores are depleted, the Fur-Fe 2ϩ dimer dissociates from the promoter, allowing the entry of RNA polymerase and subsequent transcription (22). Fur acts as a global regulator controlling not only the expression of iron acquisition...

Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in Escherichia coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5′ start of the transcript, using a single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping.

The role of the unique T-cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour-associated macrophages (TAM) and their production of microsomal prostaglandin E synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) in (Kras)-driven pancreatic tumour (KPT) progression, we crossed CD1d mice deficient in both invariant and variant NKT cells with the Kras mice. Loss of NKT cells significantly increased pancreatic intraepithelial neoplasia (PanIN) lesions and also increased 5-LOX and mPGES-1 expression in M2-type macrophages and cancer stem-like cells in pancreatic tumours. Pharmacological inhibition of mPGES-1 and 5-LOX in M2 macrophages with specific inhibitor YS-121 in KPT-CD1d mice decreased PanIN lesions and suppressed tumour growth in association with elevated levels of active CD8a cells. Hence, NKT cells regulate PC by modulating TAMs (M2) through mPGES-1 and 5-LOX; and the absence of NKT cells leads to aggressive development of PC.

Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

bRegulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA halflives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.

Tick resistant cattle could provide a potentially sustainable and environmentally sound method of controlling cattle ticks. Advances in genomics and the availability of the bovine genome sequence open up opportunities to identify useful and selectable genes controlling cattle tick resistance. Using quantitative real-time PCR and theAffymetrix bovine array platform, differences in gene expression of skin biopsies from tick resistant Bos indicus (Brahman) and tick susceptible Bos taurus (Holstein-Friesian) cattle following tick challenge were examined. We identified 138 significant differentially-expressed genes, including several immunologicallhost defence genes, extracellularmatrix proteins, and transcription factors as well as genes involved in lipid metabolism. Three key pathways, represented by genes differentially expressed in resistant Brahmans, were identified; the development of the cell-mediated immune response, structural integrity of the dermis and intracellular Ca2+ levels. Ca2+, which is implicated in host responses to microbial stimuli, may be required for the enhancement or fine-tuning of transcriptional activation of Ca2+ -dependant host defence signalling pathways.