Identification of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Rad51C: Recombinational characteristics

Unicellular green alga Chlamydomonas reinhardtii is a promising model for fundamental and biotechnological research. However, little is known about its system of homologous recombination underlying recombination repair of double-strand breaks. Sequencing of the C. reinhardtii nuclear genome has revealed many repeats, which account for a low level of nuclear homologous recombination compared to that of nonhomologous recombination. Analysis of C. reinhardtii EST and genomic libraries made it possible to reconstruct and clone the RAD51C cDNA. In this work, this cDNA was expressed, the protein product was purified, and its main biochemical activities were studied. It was shown that Rad51C of lower eukaryote C. reinhardtii is a typical member of the subfamily of higher eukaryotic Rad51-like recombination proteins.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 112–119.Original Russian Text Copyright © 2005 by Shalguev, Kaboev, Sizova, Hagemann, Lanzov.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Roles of <Emphasis Type="Italic">Saccharomyces cerevisiae RAD17</Emphasis> and <Emphasis Type="Italic">CHK1</Emphasis> checkpoint genes in the repair of double-strand breaks in cycling cells

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Δ/chk1Δ and rad17Δ/rad17Δ, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Δ/rad17Δ cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Δ/rad17Δ cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Δ/chk1Δ cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Δ/chk1Δ mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background. Nelson Bracesco and Ema C. Candreva have contributed equally to this article.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Candida albicans</Emphasis>, a major human fungal pathogen

Candida albicans is the most common human fungal pathogen (Beck-Sague and Jarvis, 1993). It is normally a harmless commensal organism. However, it is a opportunistic pathogen for some immunologically weak and immunocompromised people. It is responsible for painful mucosal infections such as the vaginitis in women and oral-pharangeal thrush in AIDS patients. In certain groups of vulnerable patients it causes severe, life-threatening bloodstream infections and it causes severe, life-threatening bloodstream infections and subsequent infections in the internal organs. There are various fascinating features of the C. albicans life cycle and biology that have made the pathogen the subject of extensive research, including its ability to grow in unicellular yeast, psudohyphal, and hyphal forms (Fig. 1A); its ability to switch between different but stable phenotypic states, and the way that it retains the ability to mate but apparently loses the ability to go through meiosis to complete the sexual cycle. This research has been greatly facilitated by the derivation of the complete C. albicans genome sequence (Braun et al., 2005), the development of a variety of molecular tools for gene manipulation, and a store of underpinning knowledge of cell biology borrowed from the distantly related model yeast Saccharomyces cerevisiae (Berman and Sudbery, 2002; Noble and Johnson, 2007). This review will provide a brief overview of the importance of C. albicans as a public health issue, the experimental tools developed to study its fascinating biology, and some examples of how these have been applied.     

Biofilm formation and adhesive/invasive properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in comparison with <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida dubliniensis and Candida albicans are closely related spp. exhibiting differences in their virulence potency. This study compared clinical isolates of C. dubliniensis with C. albicans from HIV patients with oropharyngeal candidiasis (OPC) and standard strains in power to form biofilm and their adhesive and invasive properties. Members of both spp. were able to form strong biofilms. However, SEM microscopy confirmed that C. albicans undergoes the more effective yeast-to-hyphae transition than C. dubliniensis with prevalent yeast form and limited ability to form filaments. Kinetic patterns indicated that while the first 30 min are critical for sufficient attachment to a polystyrene surface, adhesion to human carcinoma cell lines (Caco-2 and TR 146) needs additional time with maximal saturation observed at 240 min for both spp. The invasion process was tested on 3D RHE (reconstituted human epithelium) with Caco-2 or TR 146 on the collagen surface. C. albicans rapidly produced hyphae that penetrated the tissue layer, demonstrating substantive invasion within 21 h. In contrast, C. dubliniensis attached to the tissue surface and proliferated, suggesting the formation of a biofilm-like structure. After 21 h, C. dubliniensis was able to penetrate the RHE layer and invade unusually, with a cluster of the yeast cells.     

Cloning,Expression and Purification of <Emphasis Type="Italic">Microcystis viridis</Emphasis> Lectin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microcystis viridis lectin (MVL), a sugar-binding protein originally isolated from freshwater blue-green algae Microcystis viridis, has been reported to have potent anti-HIV activity. In this paper, we described the expression and purification of recombinant-MVL (R-MVL) gene in E. coli. The results demonstrated that the R-MVL in shake flask cultures was primarily expressed either in the form of inclusion bodies at 37°C or in the soluble fraction at 23 °C. Secondly, a one-step purification based on nickel-affinity chromatography was employed and 15 mg of highly purified (>95%) R-MVL from 1 l of cell cultures was yielded. The purified R-MVL was then subjected to MALDI-TOF–MS analysis for protein identification. In conclusion, for the first time, the R-MVL was successfully cloned and expressed in E. coli, which is useful for further study and large-scale cost-effective production of MVL protein.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Characterization of the <Emphasis Type="Italic">Bm61</Emphasis> of the <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.     

Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading <Emphasis Type="Italic">Candida albicans</Emphasis> TL3

A eukaryotic catechol 1,2-dioxygenase (1,2-CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity. The 1,2-CTD was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration, and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. The optimal temperature and pH were 25°C and 8.0, respectively. Substrate analysis showed that the purified enzyme was a type I catechol 1,2-dioxygenase. This is the first time that a 1,2-CTD from a eukaryote (Candida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST analysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314. We conclude that the hypothetical protein is 1,2-CTD.