The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.

The heterochromatin domain at the mat locus of Schizosaccharomyces pombe is bounded by the IR-L and IR-R barriers. A genetic screen for mutations that promote silencing beyond IR-L revealed a novel gene named epe1, encoding a conserved nuclear protein with a jmjC domain. Disruption of epe1 promotes continuous spreading of heterochromatin-associated histone modifications and Swi6 binding to chromatin across heterochromatic barriers. It also enhances position effect variegation at heterochromatic domains, suppresses mutations in silencing genes, and stabilizes the repressed epigenetic state at the mat locus. However, it does not enhance silencing establishment. Our analysis suggests that the jmjC domain is essential for Epe1 activity and that Epe1 counteracts transcriptional silencing by negatively affecting heterochromatin stability. Consistent with this proposition, the meiotic stability of established heterochromatin beyond IR-L is diminished by Epe1 activity, and overexpression of Epe1 disrupts heterochromatin through acetylation of H3-K9 and H3-K14 and methylation of H3-K4. Furthermore, overexpression of Epe1 elevates the rate of chromosome loss. We propose that Epe1 helps control chromatin organization by down-regulating the stability of epigenetic marks that govern heterochromatization.

Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both alpha and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly alpha, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between alpha and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the alpha allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39 degrees C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Delta mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity.

Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45 degrees C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45 degrees C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.

In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MATMATMATMATMATMATMATMATMATMATMAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling.

OBJECTIVE

High plantar foot pressures in association with peripheral neuropathy have been ascertained to be important risk factors for ulceration in the diabetic foot. Most studies investigating these parameters have been limited by their size and the homogeneity of study subjects. The objective of this study was therefore to ascertain the risk of ulceration associated with high foot pressures and peripheral neuropathy in a large and diverse diabetic population.

METHODS

We studied a cross-sectional group of 251 diabetic patients of Caucasian (group C) (n=121), black (group B) (n=36), and Hispanic (group H) (n=94) racial origins with an overall age of 58.5+/-12.5 years (range 20-83). There was an equal distribution of men and women across the entire study population. All patients underwent a complete medical history and lower extremity evaluation for neuropathy and foot pressures. Neuropathic parameters were dichotomized (0/1) into two high-risk variables: patients with a vibration perception threshold (VPT)>> or =25 V were categorized as HiVPT (n=132) and those with Semmes-Weinstein monofilament tests>> or =5.07 were classified as HiSWF (n=190). The mean dynamic foot pressures of three footsteps were measured using the F-scan mat system with patients walking without shoes. Maximum plantar pressures were dichotomized into a high-pressure variable (Pmax6) indicating those subjects with pressures>> or =6 kg/cm2 (n=96). A total of 99 patients had a current or prior history of ulceration at baseline.

RESULTS

Joint mobility was significantly greater in the Hispanic cohort compared with the other groups at the first metatarsal-phalangeal joint (C 67+/-23 degrees, B 69+/-23 degrees, H 82+/-23 degrees, P=0.000), while the subtalar joint mobility was reduced in the Caucasian group (C 21+/-8 degrees, B 26+/-7 degrees, H 27+/-11 degrees, P=0.000). Maximum plantar foot pressures were significantly higher in the Caucasian group (C 6.7+/-2.9 kg/cm2, B 5.7+/-2.8 kg/cm2, H 4.4+/-1.9 kg/cm2, P=0.000). Univariate logistic regression for Pmax6 on the history of ulceration yielded an odds ratio (OR) of 3.9 (P=0.000). For HiVPT, the OR was 11.7 (P=0.000), and for HiSWF the OR was 9.6 (P=0.000). Controlling for age, diabetes duration, sex, and race (all P < 0.05), multivariate logistic regression yielded the following significant associations with ulceration: Pmax6 (OR=2.1, P=0.002), HiVPT (OR=4.4, P=0.000), and HiSWF (OR=4.1, P=0.000).

CONCLUSIONS

We conclude that both high foot pressures >> or =6 kg/cm2) and neuropathy are independently associated with ulceration in a diverse diabetic population, with the latter having the greater magnitude of effect. In black and Hispanic diabetic patients especially, joint mobility and plantar pressures are less predictive of ulceration than in Caucasians.

Liver-specific and non-liver-specific methionine adenosyltransferase (MAT) are products of two genes, MATMATMATMAT expression have not been examined in animal models of alcoholic liver injury, which is the focus of the current study. After rats were fed intragastrically with ethanol and high fat for 9 wk, the mRNA level of both MAT forms doubled but only the protein level of MATMAT activity did not change, it was 32% lower after one and 68% lower after eight weekly enteral doses of lipopolysaccharide. Hepatic levels of methionine, SAM, and DNA methylation fell by approximately 40%. c-myc was hypomethylated, and its mRNA level increased. Genome-wide DNA strand break increased. Thus in the prefibrotic stage of alcoholic liver injury, there is already a switch in MAT expression, global DNA hypomethylation, increased c-myc expression, and genome-wide DNA strand break. These changes may be important in predisposing this liver disease to malignant degeneration.

We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter.

Homothallic switching of the mating-type MATa gene in Saccharomyces cerevisiae results from replacement by gene conversion of MAT-Ya DNA with Y(alpha) sequences copied from one of two unexpressed donors. MATa preferentially recombines with HML(alpha), located near the left end of chromosome III, but can use HMR(alpha), near the right chromosome end. MATa donor preference depends on a 700 bp orientation-independent cis-acting recombination enhancer, located 17 kb proximal to HML. Deletion of this element markedly reduces MATa's use of a donor inserted at any of four different locations along the leftmost 92 kb of chromosome III. This enhancer is sufficient for donor activation, since it stimulates use of the "wrong" donor, when it is inserted 7 kb proximal to HMR.

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the Mat-Ly-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the alpha mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual alpha population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (alpha-alpha) also occurs in nature as evidenced by the existence of natural diploid alphaADalpha hybrids that arose by fusion between two alpha cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, approximately 500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the alpha MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct alpha cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.

A novel thermophilic, photosynthetic bacterium, designated strain HLO8T, was isolated from a bacterial mat in a Japanese hot spring. Morphologically, the isolate was an unbranched multicellular filament with a cell diameter of 0.8-1.0 microm. The bacterium was red to reddish-brown in colour and formed a distinct red bacterial mat in the natural environment. It was able to grow photoheterotrophically under anaerobic light conditions and also chemoheterotrophically under aerobic dark conditions. Optimal growth occurred at 50 degrees C and pH 7.5-8.0. The cells contained bacteriochlorophyll (Bchl) a and gamma-carotene derivatives as photosynthetic pigments, but lacked Bchl c and chlorosomes. Cellular fatty acids in the isolate were mainly C16:0, C14:0 and C15:0. The major quinone was menaquinone-11. The DNA G+C content was 62.0 mol% (by HPLC). Phylogenetic analysis based on 16S rDNA sequencing suggested that the isolate belonged to the anoxygenic filamentous phototrophic bacteria represented by Chloroflexus aurantiacus, but was clearly distant from all members in this group (the sequence similarities between the isolate and its relatives were less than 83.8%). Based on genotypic and phenotypic data, the name Roseiflexus castenholzii gen. nov., sp. nov. is proposed for this isolate; the type strain is HLO8T (= DSM 13941T = JCM 11240T).

Chitosan nanofibers were electrospun from aqueous chitosan solution using concentrated acetic acid solution as a solvent. A uniform nanofibrous mat of average fiber diameter of 130 nm was obtained from the following optimum condition: 7% chitosan solution in aqueous 90% acetic acid solution was successfully electrospun in the electric field of 4 kV/cm. The aqueous acetic acid concentration higher than 30% was prerequisite for chitosan nanofiber formation, because more concentrated acetic acid in water progressively decreased surface tension of the chitosan solution and concomitantly increased charge density of jet without significant effect on solution viscosity. However, acetic acid solution more than 90% did not dissolve enough chitosan to make spinnable viscous concentration. Only chitosan of a molecular weight of 106,000 g/mol produced bead-free chitosan nanofibers, while low- or high-molecular-weight chitosans of 30,000 and 398,000 g/mol did not. Average fiber diameters and size distribution decreased with increasing electric field and more bead defects appeared at 5 kV/cm or more.

A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10 degrees C) to high temperature (167 degrees C). The vent fluid is characterized by high concentrations of CO2 (up to 17 mM) and Fe(II) (up to 268 microM), but there is a general paucity of H2S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had >50% sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 x 10(7) to 5.3 x 10(8) cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10(-6) and 10(-7). A pure culture was obtained from a 10(-7) dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30 degrees C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of Fe-oxidizing bacteria at Loihi. Together, these results indicate that Fe-oxidizing bacteria are common at the Loihi Seamount and probably play a major role in Fe oxidation. A review of the literature suggests that microbially mediated Fe oxidation at hydrothermal vents may be important globally.

The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.

Phototrophic microbial mat communities from 60°C and 65°C regions in the effluent channels of Mushroom and Octopus Springs (Yellowstone National Park, WY, USA) were investigated by shotgun metagenomic sequencing. Analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. Linkage of phylogenetic marker genes and functional genes showed novel chlorophototrophic bacteria belonging to uncharacterized lineages within the order Chlorobiales and within the Kingdom Chloroflexi. The latter is the first chlorophototrophic member of Kingdom Chloroflexi that lies outside the monophyletic group of chlorophototrophs of the Order Chloroflexales. Direct comparison of unassembled metagenomic sequences to genomes of representative isolates showed extensive genetic diversity, genomic rearrangements and novel physiological potential in native populations as compared with genomic references. Synechococcus spp. metagenomic sequences showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B', but synteny declined with decreasing sequence relatedness to these references. There was evidence of horizontal gene transfer among native populations, but the frequency of these events was inversely proportional to phylogenetic relatedness.

The Hippo pathway is an evolutionally conserved protein kinase cascade involved in regulating organ size in vivo and cell contact inhibition in vitro by governing cell proliferation and apoptosis. Deregulation of the Hippo pathway is linked to cancer development. Its first core kinase Warts was identified in Drosophila more than 15 years ago, but it gained much attention when other core components of the pathway were identified 8 years later. Major discoveries of the pathway were made during past several years. The core kinase components Hippo, Salvador, Warts, and Mats in the fly and Mst1/2, WW45, Lats1/2, and Mob1 in mammals phosphorylate and inactivate downstream transcriptional co-activators Yorkie in the fly, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in mammals, respectively. Phosphorylated Yorkie, YAP, and TAZ are sequestered in the cytoplasm by interaction with 14-3-3 proteins. Here we review recent progresses of this pathway by focusing on how these proteins communicate with each other and how loss of regulation results in cancers.

OBJECTIVE

To investigate the effects of 24 weeks of whole-body-vibration (WBV) training on knee-extension strength and speed of movement and on counter-movement jump performance in older women.

METHODS

A randomized, controlled trial.

METHODS

Exercise Physiology and Biomechanics Laboratory, Leuven, Belgium.

METHODS

Eighty-nine postmenopausal women, off hormone replacement therapy, aged 58 to 74, were randomly assigned to a WBV group (n=30), a resistance-training group (RES, n=30), or a control group (n=29).

METHODS

The WBV group and the RES group trained three times a week for 24 weeks. The WBV group performed unloaded static and dynamic knee-extensor exercises on a vibration platform, which provokes reflexive muscle activity. The RES group trained knee-extensors by performing dynamic leg-press and leg-extension exercises increasing from low (20 repetitions maximum (RM)) to high (8RM) resistance. The control group did not participate in any training.

METHODS

Pre-, mid- (12 weeks), and post- (24 weeks) isometric strength and dynamic strength of knee extensors were measured using a motor-driven dynamometer. Speed of movement of knee extension was assessed using an external resistance equivalent to 1%, 20%, 40%, and 60% of isometric maximum. Counter-movement jump performance was determined using a contact mat.

RESULTS

Isometric and dynamic knee extensor strength increased significantly (P<.001) in the WBV group (mean+/-standard error 15.0+/-2.1% and 16.1+/-3.1%, respectively) and the RES group (18.4+/-2.8% and 13.9+/-2.7%, respectively) after 24 weeks of training, with the training effects not significantly different between the groups (P=.558). Speed of movement of knee extension significantly increased at low resistance (1% or 20% of isometric maximum) in the WBV group only (7.4+/-1.8% and 6.3+/-2.0%, respectively) after 24 weeks of training, with no significant differences in training effect between the WBV and the RES groups (P=.391; P=.142). Counter-movement jump height enhanced significantly (P<.001) in the WBV group (19.4+/-2.8%) and the RES group (12.9+/-2.9%) after 24 weeks of training. Most of the gain in knee-extension strength and speed of movement and in counter-movement jump performance had been realized after 12 weeks of training.

CONCLUSIONS

WBV is a suitable training method and is as efficient as conventional RES training to improve knee-extension strength and speed of movement and counter-movement jump performance in older women. As previously shown in young women, it is suggested that the strength gain in older women is mainly due to the vibration stimulus and not only to the unloaded exercises performed on the WBV platform.

Mating type (MAT) genes were cloned from three members of the Gibberella/Fusarium complex that differ in reproductive mode: heterothallic G. fujikuroi, homothallic G. zeae, and asexual F. oxysporum. The G. fujikuroi MAT locus organization is typical of other heterothallic pyrenomycetes characterized to date; i.e., there are three genes at MATMATMAT idiomorphs, tightly linked on the same chromosome, interspersed with sequences unique to G. zeae. Field isolates of F. oxysporum, although asexual, have either the MATMATMAT genes are expressed and that all putative introns found in each of the four MAT genes in G. fujikuroi and F. oxysporum are removed. Apparent failure of F. oxysporum to reproduce sexually could not be attributed to mutations in the MAT genes themselves.

Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female-specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone-mediated process. In this study, we characterize the pre-1 gene, encoding a predicted G-protein-coupled receptor with sequence similarity to fungal pheromone receptors. pre-1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre-1 deletion mutants showed that loss of pre-1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Deltapre-1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Galpha subunit, GNA-1, and the Gbeta protein, GNB-1, are essential for female fertility in N. crassa. Trichogynes of Deltagna-1 and Deltagnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Deltapre-1 mat A strains. However, the female sterility defect of the Deltapre-1 mat A mutant could not be complemented by constitutive activation of gna-1, suggesting additional layers of regulation. We propose that PRE-1 is a pheromone receptor coupled to GNA-1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.

The Cryptococcus neoformans STE12alpha gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)alpha cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12alpha. The ability to form hyphae, however, was not affected by deletion of STE12alpha when convergently growing MATa strains were present. Furthermore, ste12alpha disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12alpha disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12alpha locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12alpha cells than those of mice infected with STE12alpha cells. Using reporter gene constructs, we found that STE12alpha controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.

During sporulation of Saccharomyces cerevisiae, meiosis is followed by encapsulation of haploid nuclei within multilayered spore walls. Completion of the late events of the sporulation program requires the SPS1 gene. This developmentally regulated gene, which is expressed as cells are nearing the end of meiosis, encodes a protein with homology to serine/threonine protein kinases. The catalytic domain of Sps1 is 44% identical to the kinase domain of yeast Ste20, a protein involved in the pheromone-induced signal transduction pathway. Cells of a MATa/MAT alpha sps1/sps1 strain arrest after meiosis and fail to activate genes that are normally expressed at a late time of sporulation. The mutant cells do not form refractile spores as assessed by phase-contrast microscopy and do not display the natural fluorescence and ether resistance that is characteristic of mature spores. Examination by electron microscopy reveals, however, that prospore-like compartments form in some of the mutant cells. These immature spores lack the cross-linked surface layer that surrounds wild-type spores and are more variable in size and number than are the spores of wild-type cells. Despite their inability to complete spore formation, sps1-arrested cells are able to resume mitotic growth on transfer to rich medium, generating haploid progeny. Our results suggest that the developmentally regulated Sps1 kinase is required for normal progression of transcriptional, biochemical, and morphological events during the later portion of the sporulation program.