A component of the TOR (Target Of Rapamycin) nutrient-sensing pathway plays a role in circadian rhythmicity in Neurospora crassa.

The TOR (Target of Rapamycin) pathway is a highly-conserved signaling pathway in eukaryotes that regulates cellular growth and stress responses. The cellular response to amino acids or carbon sources such as glucose requires anchoring of the TOR kinase complex to the lysosomal/vacuolar membrane by the Ragulator (mammals) or EGO (yeast) protein complex. Here we report a connection between the TOR pathway and circadian (daily) rhythmicity. The molecular mechanism of circadian rhythmicity in all eukaryotes has long been thought to be transcription/translation feedback loops (TTFLs). In the model eukaryote Neurospora crassa, a TTFL including FRQ (frequency) and WCC (white collar complex) has been intensively studied. However, it is also well-known that rhythmicity can be seen in the absence of TTFL functioning. We previously isolated uv90 as a mutation that compromises FRQ-less rhythms and also damps the circadian oscillator when FRQ is present. We have now mapped the uv90 gene and identified it as NCU05950, homologous to the TOR pathway proteins EGO1 (yeast) and LAMTOR1 (mammals), and we have named the N. crassa protein VTA (vacuolar TOR-associated protein). The protein is anchored to the outer vacuolar membrane and deletion of putative acylation sites destroys this localization as well as the protein's function in rhythmicity. A deletion of VTA is compromised in its growth responses to amino acids and glucose. We conclude that a key protein in the complex that anchors TOR to the vacuole plays a role in maintaining circadian (daily) rhythmicity. Our results establish a connection between the TOR pathway and circadian rhythms and point towards a network integrating metabolism and the circadian system.

A new mutation affecting FRQ-less rhythms in the circadian system of Neurospora crassa.

We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq(10) knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.

Molecular characterization of a Squalene epoxidase gene in dermatophyte pathogen Trichophyton tonsurans.

BACKGROUND: Trichophyton tonsurans is one of the dermatophyte fungi which invades the skin and hair of human. Several properties of this fungus have been investigated so far. However a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the Squalene epoxidase gene which is related to synthesis of ergosterol in this fungus. METHODS: Pairs of 23 and 24 nucleotides primers were designed from highly conserved regions of the similar genes in other fungi. Mentioned primers were utilized in PCR by using isolated genomic DNA of T. tonsurans whereas the PCR fragments were then sequenced. RESULTS AND CONCLUSION: Nucleotides (n = 558) have been sequenced from this new gene which encodes a polypeptide with 186 amino acids. Sequences comparison in gene data banks (NCBI, NIH) for this part of DNA and its deduced amino acid revealed significant homology with members of the eukaryotic Squalene epoxidase.

A tripartite toxin-antitoxin module induced by quorum sensing is associated with the persistence phenotype in Streptococcus mutans.

The oral pathogen Streptococcus mutans communicates using a canonical Gram-positive quorum sensing system, CSP-ComDE. The CSP pheromone already known to be involved in the development of genetic competence positively influences the formation of persisters, dormant variants of regular cells that are highly tolerant to antimicrobial therapy. It is now believed that the persistence phenotype is the end result of a stochastic switch in the expression of toxin-antitoxin (TA) modules. TAs consist of a pair of genes that encode two components, a stable toxin and its cognate labile antitoxin. Transcription analyses revealed that three core genes encoding a putative TA system, called SmuATR, were members of the S. mutans CSP regulon. We hypothesized that S. mutans is using its CSP-ComDE system as a deterministic mechanism for persister formation through the activation of smuATR locus. We showed here that the SmuATR system constitutes a novel tripartite type II TA system in which the smuA and smuT genes encode an antitoxin and a toxin, respectively, while SmuR is a transcriptional repressor involved in the autoregulation of the operon. Ectopic expression of SmuA - SmuT is associated with the CSP-inducible persistence phenotype. In contrast, overexpression of SmuT alone is bactericidal and causes membrane permeabilization. To our knowledge, SmuATR is the first functional chromosomal tripartite TA system shown to be induced by the bacterial quorum sensing system and involved in persister formation.

PRD-1, a Component of the Circadian System of Neurospora crassa, Is a Member of the DEAD-box RNA Helicase Family.

The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2 Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1-GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases (prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s).

Phylogenetic relationship of Salmonella enterica strains in Tehran, Iran, using 16S rRNA and gyrB gene sequences.

INTRODUCTION: We assessed whether 16S rDNA and gyrB gene sequences, alone or combined, were suitable for determining the phylogenetic relationship among Salmonella enterica strains isolated from Tehran, Iran. Patients over five years of age enrolled in an acute diarrheal surveillance project in Tehran province between May 2004 and October 2006 were selected as our study group. METHODOLOGY: 16S ribosomal DNA (rDNA) and gyrB genes from 40 Salmonella isolates obtained from patients with acute diarrhea were sequenced and the data was used to generate phylogenetic trees that facilitated isolate comparison. RESULTS: Salmonella strains clustered into five to seven phylogenetic groups, dependent on analysis of 16S rDNA (1546 bp), gyrB (1256 bp) or a combination of the two genes. By 16S rDNA sequence analysis, only strains of Salmonella enterica serovar Typhi ( S. Typhi) clustered exclusively together. gyrB sequences permitted clustering of all the S. Typhi and S. Paratyphi A isolates, and clustering of S. Enteritidis into two separate but exclusive groups. Concatenation of the two data sets did not significantly improve the resolution of the strains compared to the gyrB gene. None of the analyses completely resolved S. enterica Paratyphi B and C into mutually exclusive groups. CONCLUSION: Sequencing of gyrB represents a potentially useful tool for determining the phylogenetic relationship of S. enterica strains in Tehran, Iran. Genetic analysis of the 16S rRNA gene alone or in combination with gyrB did not increase the resolution between serotypes of S. enterica. We speculate that inclusion of additional genetic markers would improve the sensitivity of the analysis.