Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors.

The genus Pneumocystis encompasses fungal species that colonize mammals' lungs with host specificity. Should the host immune system weaken, the fungal species can cause severe pneumonia. The life cycle of these pathogens is poorly known, mainly because an in vitro culture method has not been established. Both asexual and sexual cycles would occur. Trophic cells, the predominant forms during infection, could multiply asexually but also enter into a sexual cycle. Comparative genomics revealed a single mating type locus, including plus and minus genes, suggesting that primary homothallism involving self-fertility of each strain is the mode of reproduction of Pneumocystis species. We identified and analyzed the expression of the mam2 and map3 genes encoding the receptors for plus and minus pheromones using reverse transcriptase PCR, in both infected mice and bronchoalveolar lavage fluid samples from patients with Pneumocystis pneumonia. Both receptors were most often concomitantly expressed during infection, revealing that both pheromone-receptor systems are involved in the sexual cycle. The map3 transcripts were subject to alternative splicing. Using immunostaining, we investigated the presence of the pheromone receptors at the surfaces of Pneumocystis cells from a patient. The staining tools were first assessed in Saccharomyces cerevisiae displaying the Pneumocystis receptors at their cellular surface. Both receptors were present at the surfaces of the vast majority of the cells that were likely trophic forms. The receptors might have a role in mate recognition and/or postfertilization events. Their presence at the cell surface might facilitate outbreeding versus inbreeding of self-fertile strains.IMPORTANCE The fungi belonging to the genus Pneumocystis may cause severe pneumonia in immunocompromised humans, a disease that can be fatal if not treated. This disease is nowadays one of the most frequent invasive fungal infections worldwide. Whole-genome sequencing revealed that the sexuality of these fungi involves a single partner that can self-fertilize. Here, we report that two receptors recognizing specifically excreted pheromones are involved in this self-fertility within infected human lungs. Using fluorescent antibodies binding specifically to these receptors, we observed that most often, the fungal cells display both receptors at their surface. These pheromone-receptor systems might play a role in mate recognition and/or postfertilization events. They constitute an integral part of the Pneumocystis obligate sexuality within human lungs, a cycle that is necessary for the dissemination of the fungus to new individuals.

Organic substrate diffusibility governs microbial community composition, nutrient removal performance and kinetics of granulation of aerobic granular sludge.

Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20-40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation - ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW - composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality through Primary Homothallism within Host Lungs.

Fungi of the genus Pneumocystis are obligate parasites that colonize mammals' lungs and are host species specific. Pneumocystis jirovecii and Pneumocystis carinii infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture in vitro It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type (MAT) locus encompassing plus and minus genes (matMc, matMi, and matPi; Almeida et al., mBio 6:e02250-14, 2015). Thus, each strain would be capable of sexual reproduction alone (self-fertility). However, this is a working hypothesis derived from computational analyses that is, in addition, based on the genome sequences of single isolates. Here, we tested this hypothesis in the wet laboratory. The function of the P. jirovecii and P. carinii matMc genes was ascertained by restoration of sporulation in the corresponding mutant of fission yeast. Using PCR, we found the same single MAT locus in all P. jirovecii isolates and showed that all three MAT genes are often concomitantly expressed during pneumonia. Extensive homology searches did not identify other types of MAT transcription factors in the genomes or cis-acting motifs flanking the MAT locus that could have been involved in MAT switching or silencing. Our observations suggest that Pneumocystis sexuality through primary homothallism is obligate within host lungs to complete the cell cycle, i.e., produce asci necessary for airborne transmission to new hosts.IMPORTANCE Fungi of the genus Pneumocystis colonize the lungs of mammals. In immunosuppressed human hosts, Pneumocystis jirovecii may cause severe pneumonia that can be fatal. This disease is one of the most frequent life-threatening invasive fungal infections in humans. The analysis of the genome sequences of these uncultivable pathogens suggested that their sexual reproduction involves a single partner (self-fertilization). Here, we report laboratory experiments that support this hypothesis. The function of the three genes responsible for sexual differentiation was ascertained by the restoration of sexual reproduction in the corresponding mutant of another fungus. As predicted by self-fertilization, all P. jirovecii isolates harbored the same three genes that were often concomitantly expressed within human lungs during infection. Our observations suggest that the sexuality of these pathogens relies on the self-fertility of each isolate and is obligate within host lungs to complete the cell cycle and allow dissemination of the fungus to new hosts.

Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

Soil fungal communities of grasslands are environmentally structured at a regional scale in the Alps.

Studying patterns of species distributions along elevation gradients is frequently used to identify the primary factors that determine the distribution, diversity and assembly of species. However, despite their crucial role in ecosystem functioning, our understanding of the distribution of below-ground fungi is still limited, calling for more comprehensive studies of fungal biogeography along environmental gradients at various scales (from regional to global). Here, we investigated the richness of taxa of soil fungi and their phylogenetic diversity across a wide range of grassland types along a 2800 m elevation gradient at a large number of sites (213), stratified across a region of the Western Swiss Alps (700 km(2)). We used 454 pyrosequencing to obtain fungal sequences that were clustered into operational taxonomic units (OTUs). The OTU diversity-area relationship revealed uneven distribution of fungal taxa across the study area (i.e. not all taxa are everywhere) and fine-scale spatial clustering. Fungal richness and phylogenetic diversity were found to be higher in lower temperatures and higher moisture conditions. Climatic and soil characteristics as well as plant community composition were related to OTU alpha, beta and phylogenetic diversity, with distinct fungal lineages suggesting distinct ecological tolerances. Soil fungi, thus, show lineage-specific biogeographic patterns, even at a regional scale, and follow environmental determinism, mediated by interactions with plants.

Can hyperbaric oxygenation therapy (HOT) modify the blood testosterone concentration?

INTRODUCTION: Testosterone has a modulating effect on inflammatory and healing processes. In this study, we evaluate whether hyperbaric oxygenation therapy (HOT) modifies the blood concentration of total testosterone (TT) in patients treated for different pathologies. MATERIALS AND METHODS: Fourteen male patients (23-72 years old) were treated with 90-min HOT sessions (range 4 to 23 sessions) as an adjuvant to the following conditions: leg fractures, osteonecrosis, diabetic foot, firearm injuries, complicated arthroprosthesis and underwater diving embolism. As controls, six healthy male volunteers (37-51 years old) were subjected to 10 HOT sessions. Testosterone plasma levels were determined immediately before the first HOT session and the day after the last session. RESULTS: At the end of treatment, 12 patients fully recovered and 2 (diabetic foot patients) showed a marked improvement. Testosterone significantly increased after hyperbaric oxygenation therapy in both patients and controls (ANOVA, p<0.004). DISCUSSION: We conclude that hyperbaric oxygenation therapy increases the blood concentration of total testosterone in patients as well as in healthy men. This finding raises new questions and indicates the need to investigate the causes of this increase and its therapeutic significance. Since testosterone modulates inflammation and healing processes, it is possible that hormonal changes are the mechanisms affected by hyperbaric oxygenation therapy.

The elusive roles of bacterial glutathione S-transferases: new lessons from genomes.

Glutathione S-transferases constitute a large family of enzymes which catalyze the addition of glutathione to endogenous or xenobiotic, often toxic electrophilic chemicals. Eukaryotic glutathione S-transferases usually promote the inactivation, degradation or excretion of a wide range of compounds by formation of the corresponding glutathione conjugates. In bacteria, by contrast, the few glutathione S-transferases for which substrates are known, such as dichloromethane dehalogenase, 1,2-dichloroepoxyethane epoxidase and tetrachlorohydroquinone reductase, are catabolic enzymes with an essential role for growth on recalcitrant chemicals. Glutathione S-transferase genes have also been found in bacterial operons and gene clusters involved in the degradation of aromatic compounds. Information from bacterial genome sequencing projects now suggests that glutathione S-transferases are present in large numbers in proteobacteria. In particular, the genomes of three Pseudomonas species each include at least ten different glutathione S-transferase genes. Several of the corresponding proteins define new classes of the glutathione S-transferase family and may also have novel functions that remain to be elucidated.

mmsearch: a motif arrangement language and search program.

UNLABELLED: This paper presents a language for describing arrangements of motifs in biological sequences, and a program that uses the language to find the arrangements in motif match databases. The program does not by itself search for the constituent motifs, and is thus independent of how they are detected, which allows it to use motif match data of various origins. AVAILABILITY: The program can be tested online at http://hits.isb-sib.ch and the distribution is available from ftp://ftp.isrec.isb-sib.ch/pub/software/unix/mmsearch-1.0.tar.gz CONTACT: Thomas.Junier@isrec.unil.ch SUPPLEMENTARY INFORMATION: The full documentation about mmsearchis available from http://hits.isb-sib.ch/~tjunier/mmsearch/doc.

Making sense of score statistics for sequence alignments.

The search for similarity between two biological sequences lies at the core of many applications in bioinformatics. This paper aims to highlight a few of the principles that should be kept in mind when evaluating the statistical significance of alignments between sequences. The extreme value distribution is first introduced, which in most cases describes the distribution of alignment scores between a query and a database. The effects of the similarity matrix and gap penalty values on the score distribution are then examined, and it is shown that the alignment statistics can undergo an abrupt phase transition. A few types of random sequence databases used in the estimation of statistical significance are presented, and the statistics employed by the BLAST, FASTA and PRSS programs are compared. Finally the different strategies used to assess the statistical significance of the matches produced by profiles and hidden Markov models are presented.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

trEST, trGEN and Hits: access to databases of predicted protein sequences.

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits. isb-sib.ch).

Dotlet: diagonal plots in a web browser.

UNLABELLED: Dotlet is a program for comparing sequences by the diagonal plot method. It is designed to be platform-independent and to run in a Web browser, thus enabling the majority of researchers to use it. AVAILABILITY: The applet can be tested at http://www.isrec.isb-sib.ch/java/dotlet/ Dotlet.html, and the source code is available upon request. CONTACT: Thomas. Junier Marco.Pagni @isrec.unil.ch SUPPLEMENTARY: The full documentation about d o t l e t is available from the above URL.

InterPro--an integrated documentation resource for protein families, domains and functional sites.

MOTIVATION: InterPro is a new integrated documentation resource for protein families, domains and functional sites, developed initially as a means of rationalising the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. RESULTS: Merged annotations from PRINTS, PROSITE and Pfam form the InterPro core. Each combined InterPro entry includes functional descriptions and literature references, and links are made back to the relevant parent database(s), allowing users to see at a glance whether a particular family or domain has associated patterns, profiles, fingerprints, etc. Merged and individual entries (i.e. those that have no counterpart in the companion resources) are assigned unique accession numbers. Release 1.2 of InterPro (June 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification (PTMs) encoded by 6581 different regular expressions, profiles, fingerprints and Hidden Markov Models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1000000 hits from 264333 different proteins out of 384572 in SWISS-PROT and TrEMBL).

Genetic and physical maps of the Bacillus subtilis chromosome.

Sequencing of the complete Bacillus subtilis chromosome revealed the presence of approximately 4100 genes, 1000 of which were previously identified and mapped by classical genetic crosses. Comparison of these experimentally determined positions to those derived from the nucleotide sequence showed discrepancies reaching up to 24 degrees (approximately 280 kb). The size of these discrepancies as a function of their position along the chromosome is not random but, apparently, reveals some periodicity. Our analyses demonstrate that the discrepancies can be accounted for by inaccurate positioning of the early reference markers with respect to which all subsequently identified loci were mapped by transduction and transformation. We conclude (i) that specific DNA sequences, such as recombination hotspots or presence of heterologous DNA, had no detectable effect on the results obtained by classical mapping, and (ii) that PBS1 transduction appears to be an accurate and unbiased mapping method in B. subtilis.

Teichuronic acid operon of Bacillus subtilis 168.

Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP-glucuronate. Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls. The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription. Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer. Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.

The relationship between kinetics of substrate-limited transitions and steady-state growth in continuous cultures of Aquaspirillum autotrophicum limited by pyruvate.

Heterotrophic growth at steady state and during transient states caused by the sudden change of the concentration of the limiting factor in the feed medium was investigated experimentally for continuous cultures of Aquaspirillum autotrophicum limited by pyruvate. A model for describing the growth at steady state was selected from three unstructured models after statistical tests of the data. This model postulates that the growth yield increases linearly with the growth rate. Growth during transitions where the substrate remained limiting at all times was fitted with first-order kinetics. Theoretical predictions of these kinetics were derived from the unstructured models used to describe steady state. The predicted rate coefficients of the transients were compared to the experimental coefficients. It appeared that the model which best described steady-state growth also provided the best predictions for growth during the transient state. It is a widespread opinion that unstructured models are adequate to describe growth under steady-state conditions but not to predict transitions in continuous culture. However, for the particular case studied here, no higher degree of complexity was required to describe transitions, provided the growth of the culture was always limited by the substrate.

Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index.

A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.

Linear growth and poly(beta-hydroxybutyrate) synthesis in response to pulse-wise addition of the growth-limiting substrate to steady-state heterotrophic continuous cultures of Aquaspirillum autotrophicum.

Heterotrophic pyruvate-limited steady-state continuous cultures of the bacterium Aquaspirillum autotrophicum were perturbed with a pulse injection of a small volume of concentrated pyruvate solution. These cultures exhibited an instantaneous change in the growth dynamics, turning from steady state to apparently linear growth. These transient growth-responses had no lag phase and were clearly distinct from unlimited exponential growth according to the initial rates of increase of biomass and substrate disappearance kinetics. A linear accumulation with time of poly(beta-hydroxybutyrate) was observed within the cells. Slopes of these linear responses were negatively correlated with the dilution rate. Physiological bases of linear growth are discussed in the light of the models of H. E. Kubitschek. Poly(beta-hydroxybutyrate) synthesis in the absence of exogenous limitation may serve to protect the cells against a transient metabolic overflow.