Receptors and Other Signaling Proteins Required for Serotonin Control of Locomotion in Caenorhabditis elegans

A better understanding of the molecular mechanisms of signaling by the neurotransmitter serotonin is required to assess the hypothesis that defects in serotonin signaling underlie depression in humans. Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion, providing a genetic system to analyze serotonin signaling. From large-scale genetic screens we identified 36 mutants of C. elegans in which serotonin fails to have its normal effect of slowing locomotion, and we molecularly identified eight genes affected by 19 of the mutations. Two of the genes encode the serotonin-gated ion channel MOD-1 and the G-protein-coupled serotonin receptor SER-4. mod-1 is expressed in the neurons and muscles that directly control locomotion, while ser-4 is expressed in an almost entirely non-overlapping set of sensory and interneurons. The cells expressing the two receptors are largely not direct postsynaptic targets of serotonergic neurons. We analyzed animals lacking or overexpressing the receptors in various combinations using several assays for serotonin response. We found that the two receptors act in parallel to affect locomotion. Our results show that serotonin functions as an extrasynaptic signal that independently activates multiple receptors at a distance from its release sites and identify at least six additional proteins that appear to act with serotonin receptors to mediate serotonin response.     

Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.     

Genome sequence of Phaeobacter daeponensis type strain (DSM 23529T), a facultatively anaerobic bacterium isolated from marine sediment,and emendation of Phaeobacter daeponensis

TF-218^T is the type strain of the species Phaeobacter daeponensis Yoon et al. 2007, a facultatively anaerobic Phaeobacter species isolated from tidal flats. Here we describe the draft genome sequence and annotation of this bacterium together with previously unreported aspects of its phenotype. We analyzed the genome for genes involved in secondary metabolite production and its anaerobic lifestyle, which have also been described for its closest relative Phaeobacter caeruleus. The 4,642,596 bp long genome of strain TF-218^T contains 4,310 protein-coding genes and 78 RNA genes including four rRNA operons and consists of five replicons: one chromosome and four extrachromosomal elements with sizes of 276 kb, 174 kb, 117 kb and 90 kb. Genome analysis showed that TF-218^T possesses all of the genes for indigoidine biosynthesis, and on specific media the strain showed a blue pigmentation. We also found genes for dissimilatory nitrate reduction, gene-transfer agents, NRPS/ PKS genes and signaling systems homologous to the LuxR/I system.     

Too Much of a Good Thing: The Unique and Repeated Paths Toward Copper Adaptation

Copper is a micronutrient essential for growth due to its role as a cofactor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multilayered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1-1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1-1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient.     

Complete genome sequence of Streptococcus agalactiae strain SA20-06, a fish pathogen associated to meningoencephalitis outbreaks

Streptococcus agalactiae (Lancefield group B; GBS) is the causative agent of meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. Meningoencephalitis is a major health problem for tilapia farming and is responsible for high economic losses worldwide. Despite its importance, the genomic characteristics and the main molecular mechanisms involved in virulence of S. agalactiae isolated from fish are still poorly understood. Here, we present the genomic features of the 1,820,886 bp long complete genome sequence of S. agalactiae SA20-06 isolated from a meningoencephalitis outbreak in Nile tilapia (Oreochromis niloticus) from Brazil, and its annotation, consisting of 1,710 protein-coding genes (excluding pseudogenes), 7 rRNA operons, 79 tRNA genes and 62 pseudogenes.     

Genome sequence of the phage-gene rich marine Phaeobacter arcticus type strain DSM 23566T

Phaeobacter arcticus Zhang et al. 2008 belongs to the marine Roseobacter clade whose members are phylogenetically and physiologically diverse. In contrast to the type species of this genus, Phaeobacter gallaeciensis, which is well characterized, relatively little is known about the characteristics of P. arcticus. Here, we describe the features of this organism including the annotated high-quality draft genome sequence and highlight some particular traits. The 5,049,232 bp long genome with its 4,828 protein-coding and 81 RNA genes consists of one chromosome and five extrachromosomal elements. Prophage sequences identified via PHAST constitute nearly 5% of the bacterial chromosome and included a potential Mu-like phage as well as a gene-transfer agent (GTA). In addition, the genome of strain DSM 23566^T encodes all of the genes necessary for assimilatory nitrate reduction. Phylogenetic analysis and intergenomic distances indicate that the classification of the species might need to be reconsidered.     

A Genome-Wide RNAi Screen for Enhancers of par Mutants Reveals New Contributors to Early Embryonic Polarity in Caenorhabditis elegans

The par genes of Caenorhabditis elegans are essential for establishment and maintenance of early embryo polarity and their homologs in other organisms are crucial polarity regulators in diverse cell types. Forward genetic screens and simple RNAi depletion screens have identified additional conserved regulators of polarity in C. elegans; genes with redundant functions, however, will be missed by these approaches. To identify such genes, we have performed a genome-wide RNAi screen for enhancers of lethality in conditional par-1 and par-4 mutants. We have identified 18 genes for which depletion is synthetically lethal with par-1 or par-4, or both, but produces little embryo lethality in wild type. Fifteen of the 18 genes identified in our screen are not previously known to function in C. elegans embryo polarity and 11 of them also increase lethality in a par-2 mutant. Among the strongest synthetic lethal genes, polarity defects are more apparent in par-2 early embryos than in par-1 or par-4, except for strd-1(RNAi), which enhances early polarity phenotypes in all three mutants. One strong enhancer of par-1 and par-2 lethality, F25B5.2, corresponds to nop-1, a regulator of actomyosin contractility for which the molecular identity was previously unknown. Other putative polarity enhancers identified in our screen encode cytoskeletal and membrane proteins, kinases, chaperones, and sumoylation and deubiquitylation proteins. Further studies of these genes should give mechanistic insight into pathways regulating establishment and maintenance of cell polarity.     

Genome analysis of Desulfotomaculum gibsoniae strain GrollT a highly versatile Gram-positive sulfate-reducing bacterium

Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H₂ + CO₂ and sulfate and slowly acetogenically with H₂ + CO_2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain Groll^T together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO₂ fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes.     

Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos

Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)–treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L–like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease–causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms.     

The Genomic Blueprint of Salmonella enterica subspecies enterica serovar Typhi P-stx-12

Salmonella enterica subspecies enterica serovar Typhi is a rod-shaped, Gram-negative, facultatively anaerobic bacterium. It belongs to the family Enterobacteriaceae in the class Gammaproteobacteria, and has the capability of residing in the human gallbladder by forming a biofilm and hence causing the person to become a typhoid carrier. Here we present the complete genome of Salmonella enterica subspecies enterica serotype Typhi strain P-stx-12, which was isolated from a chronic carrier in Varanasi, India. The complete genome comprises a 4,768,352 bp chromosome with a total of 98 RNA genes, 4,691 protein-coding genes and a 181,431 bp plasmid. Genome analysis revealed that the organism is closely related to Salmonella enterica serovar Typhi strain Ty2 and Salmonella enterica serovar Typhi strain CT18, although their genome structure is slightly different.     

Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution of Drosophila centrosomin

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins.     

Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.     

Non-contiguous finished genome sequence and description of Kurthia senegalensis sp. nov.

Kurthia senegalensis strain JC8E^T sp. nov. is the type strain of K. senegalensis sp. nov., a new species within the genus Kurthia. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. K. senegalensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,975,103 bp long genome contains 2,889 protein-coding genes and 83 RNA genes, including between 4 and 6 rRNA genes.     

Genome sequence of Phaeobacter caeruleus type strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564^T was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.     

Genome of the marine alphaproteobacterium Hoeflea phototrophica type strain (DFL-43T)

Hoeflea phototrophica Biebl et al. 2006 is a member of the family Phyllobacteriaceae in the order Rhizobiales, which is thus far only partially characterized at the genome level. This marine bacterium contains the photosynthesis reaction-center genes pufL and pufM and is of interest because it lives in close association with toxic dinoflagellates such as Prorocentrum lima. The 4,467,792 bp genome (permanent draft sequence) with its 4,296 protein-coding and 69 RNA genes is a part of the Marine Microbial Initiative.     

Genome sequence of the exopolysaccharide-producing Salipiger mucosus type strain (DSM 16094T), a moderately halophilic member of the Roseobacter clade

Salipiger mucosus Martínez-Cànovas et al. 2004 is the type species of the genus Salipiger, a moderately halophilic and exopolysaccharide-producing representative of the Roseobacter lineage within the alphaproteobacterial family Rhodobacteraceae. Members of this family were shown to be the most abundant bacteria especially in coastal and polar waters, but were also found in microbial mats and sediments. Here we describe the features of the S. mucosus strain DSM 16094^T together with its genome sequence and annotation. The 5,689,389-bp genome sequence consists of one chromosome and several extrachromosomal elements. It contains 5,650 protein-coding genes and 95 RNA genes. The genome of S. mucosus DSM 16094^T was sequenced as part of the activities of the Transregional Collaborative Research Center 51 (TRR51) funded by the German Research Foundation (DFG).