Hormonema schizolunatum, a new species of dothideaceous black yeasts from phyllosphere

Two black yeast isolates from plants from the Canary Islands (Spain) are described and illustrated. Absence of Woronin bodies at simple septal pores, local coralloid terminal hyphal cells, indeterminate thallus maturation, the presence of budding cells and local conversion to meristematic growth all indicate a relationship to the Dothideaceae (Dothideales, Ascomycota). Morphological properties were consistent with the genus Hormonema Lagerberg & Melin, as defined by presence of percurrent conidiogenous loci alongside undifferentiated hyphae, and results of PCR-ribotyping supported this classification. The isolates were judged to belong to a hitherto undescribed species, characterized in particular by curved conidia soon developing transverse septa. The physiological profile of this species is also described.     

Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis

Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.     

Evolution of the Spindlin Gene in Birds: Independent Cessation of the Recombination of Sex Chromosomes at the Spindlin Locus in Neognathous Birds and Tinamous, a Palaeognathous Avian Family

Tinamous (Aves, Palaeognathae, Tinamiformes) are primitive birds, generally considered to be the sister group to the ratites. Tinamous possess a W sex-chromosome, intermediate in heterochromatization between the largely euchromatic W chromosome of the ratites and the highly condensed W chromosome of the neognathous birds. Of the four genes which are known to have diverged copies on the neognathous avian W and Z chromosome (ATP5A1, CHD1, PKC and SPIN) only the spindlin gene has W- and Z-chromosomal forms in the tinamiformes. This paper describes experiments which show that the sequences of these forms are more similar to each other and to the homologous undifferentiated spindlin gene sequences in the ratite genome than to the W or Z forms of the spindlin gene in other, neognathous species. This suggests that cessation of recombination at the spindlin locus of the ancestral W and Z chromosomes of the paleognathous tinamiformes and the neognathous avian species were independent events.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): Cloning,characterization and relation to postharvest chilling stress resistance

A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.     

Detecting network communities: an application to phylogenetic analysis

This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis.     

Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber ( Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg²⁺. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg²⁺. The NR activation state, i.e. activity in the presence of Mg²⁺ relative to activity in the absence of Mg²⁺, increased with light intensity up to 400 μmol m⁻² s⁻¹ PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg²⁺. The ATP-inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5'-AMP. These results support the view that inactivation/activation of cucumber-leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5'-AMP was also observed when activity was assayed in the absence of Mg²⁺, thus indicating that 5'-AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg²⁺ and in the −Mg²⁺ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.     

Genetic diversity and differentiation of guanaco populations from Argentina inferred from microsatellite data

Genotype data from 14 microsatellite markers were used to assess the genetic diversity and differentiation of four guanaco populations from Argentine Patagonia. These animals were recently captured in the wild and maintained in semi-captivity for fibre production. Considerable genetic diversity in these populations was suggested by the finding of a total of 162 alleles, an average mean number of alleles per locus ranging from 6.50 to 8.19, and H(e) values ranging from 0.66 to 0.74. Assessment of population differentiation showed moderate but significant values of F(ST)=0.071 (P=0.000) and R(ST)=0.083 (P=0.000). An amova test showed that the genetic variation among populations was 5.6% while within populations it was 94.4%. A number of 6.6 migrants per generation may support these results. Unambiguous individual assignment to original populations was obtained for the Pilcaniyeu, Las Heras and La Esperanza populations. The erroneous assignment of 18.75% Rio Mayo individuals to the Las Heras population can be explained by the low genetic differentiation found between these two populations. Thirty-nine of 56 loci per population combinations were in Hardy--Weinberg disequilibrium because of guanaco heterozygote deficiency, which may be explained by population subdivision. The high level of genetic diversity of the guanacos analysed here indicates that the Patagonian guanaco constitutes an important genetic resource for conservation or economic utilization programmes.