A role for MGA2, but not SPT23, in activation of transcription of ERG1 in Saccharomyces cerevisiae.

The SaccharomycescerevisiaeMGA2 gene encodes an important regulator of unsaturated fatty acid production, by controlling transcription and mRNA stability of OLE1, the gene encoding the Δ9 fatty acid desaturase. Lipid composition studies indicated that the mga2Δ strain contains elevated relative amounts of squalene when compared to wild-type cells. The deletion of the MGA2 homologue SPT23 did not impact squalene levels. To explore the role of MGA2 in the regulation of sterol synthesis, the transcription of the ERG1 gene, which encodes squalene epoxidase, was studied using an ERG1 promoter-lacZ reporter gene construct. We report here that in addition to MGA2's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. Mga2p was found to be controlled by a novel regulator in its activation of ERG1, as neither unsaturated fatty acids nor cobalt affected ERG1 expression, as had previously been shown for Mga2p's regulation of OLE1. Further, response to miconazole treatment, which inhibits production of ergosterol at a later step in the sterol biosynthetic pathway and results in up-regulation of several genes in ergosterol synthesis, was not affected in the mga2Δ mutant. In each case, the spt23Δ mutant strain shows similar ERG1 expression to wild-type cells, while the mga2Δ/spt23Δ strain shows reduced ERG1 expression, comparable to the mga2Δ, suggesting that the role of regulation of ERG1 transcription is unique to Mga2p.

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.

Arabidopsis thaliana expresses two functional isoforms of Arvp, a protein involved in the regulation of cellular lipid homeostasis.

Arv1p is involved in the regulation of cellular lipid homeostasis in the yeast Saccharomyces cerevisiae. Here, we report the characterization of the two Arabidopsis thaliana ARV genes and the encoded proteins, AtArv1p and AtArv2p. The functional identity of AtArv1p and AtArv2p was demonstrated by complementation of the thermosensitive phenotype of the arv1Delta yeast mutant strain YJN1756. Both A. thaliana proteins contain the bipartite Arv1 homology domain (AHD), which consists of an NH(2)-terminal cysteine-rich subdomain with a putative zinc-binding motif followed by a C-terminal subdomain of 33 amino acids. Removal of the cysteine-rich subdomain has no effect on Arvp activity, whereas the presence of the C-terminal subdomain of the AHD is critical for Arvp function. Localization experiments of AtArv1p and AtArv2p tagged with green fluorescent protein (GFP) and expressed in onion epidermal cells demonstrated that both proteins are exclusively targeted to the endoplasmic reticulum. Analysis of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants carrying chimeric ARV1::GUS and ARV2::GUS genes showed that ARV gene promoters direct largely overlapping patterns of expression that are restricted to tissues in which cells are actively dividing or expanding. The results of this study support the notion that plants, yeast and mammals share common molecular mechanisms regulating intracellular lipid homeostasis.

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn.

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites.

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids.

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Cluster J mycobacteriophages: intron splicing in capsid and tail genes.

Bacteriophages isolated on Mycobacterium smegmatis mc(2)155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.

Characterizing sterol defect suppressors uncovers a novel transcriptional signaling pathway regulating zymosterol biosynthesis.

erg26-1ts cells harbor defects in the 4alpha-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1ts ets1-1 and erg26-1ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 ERG7 at their loci in erg26-1ts ets1-1 and erg26-1ts and ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.

Sterol-dependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae.

We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702-12711). We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae. Using [(3)H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [(3)H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [(3)H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells.

Yeast cells lacking the ARV1 gene harbor defects in sphingolipid metabolism. Complementation by human ARV1.

arv1Delta mutant cells have an altered sterol distribution within cell membranes (Tinkelenberg, A.H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M., and Sturley, S. L. (2000) J. Biol. Chem. 275, 40667-40670), and thus it has been suggested that Arv1p may be involved in the trafficking of sterol in the yeast Saccharomyces cerevisiae and also in humans. Here we present data showing that arv1Delta mutants also harbor defects in sphingolipid metabolism. [(3)H]inositol and [(3)H]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steady-state levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1Delta cells harbored defects in the rates of biosynthesis and steady-state levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1Delta cells. Moreover, these same studies demonstrated that arv1Delta cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1Delta cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1Delta cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1Delta cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.