bluuebunny/crossref_metadata_2025 huggingface dataset:
A spectrum of gene regulatory phenomena at mammalian β-globin gene lociThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.
Authors: Fromm George , Bulger Michael
Published: November 2009

The β-globin gene cluster in mammals, consisting of a set of erythroid-specific, developmentally activated, and (or) silenced genes, has long presented a model system for the investigation of gene regulation. As the number and complexity of models of gene activation and repression have expanded, so too has the complexity of phenomena associated with the regulation of the β-globin genes. Models for expression from within the locus must account for local (promoter-proximal), distal (enhancer-mediated), and domain-wide components of the regulatory pathways that proceed through mammalian development and erythroid differentiation. In this review, we provide an overview of transcriptional activation, silencing, chromatin structure, and the function of distal regulatory elements involved in the normal developmental regulation of β-globin gene expression.
H3 phosphorylation: dual role in mitosis and interphaseThis paper is one of a selection of papers published in this Special Issue entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference and has undergone the Journal’s usual peer review process.
Authors: Pérez-Cadahía Beatriz , Drobic Bojan , Davie James R.
Published: November 2009

Chromatin condensation and subsequent decondensation are processes required for proper execution of various cellular events. During mitosis, chromatin compaction is at its highest, whereas relaxation of chromatin is necessary for DNA replication, repair, recombination, and gene transcription. Since histone proteins are directly complexed with DNA in the form of a nucleosome, great emphasis is put on deciphering histone post-translational modifications that control the chromatin condensation state. Histone H3 phosphorylation is a mark present in mitosis, where chromatin condensation is necessary, and in transcriptional activation of genes, when chromatin needs to be decondensed. There are four characterized phospho residues within the H3 N-terminal tail during mitosis: Thr3, Ser10, Thr11, and Ser28. Interestingly, H3 phosphorylated at Ser10, Thr11, and Ser28 has been observed on genomic regions of transcriptionally active genes. Therefore, H3 phosphorylation is involved in processes requiring opposing chromatin states. The level of H3 phosphorylation is mediated by opposing actions of specific kinases and phosphatases during mitosis and gene transcription. The cellular contexts under which specific residues on H3 are phosphorylated in mitosis and interphase are known to some extent. However, the functional consequences of H3 phosphorylation are still unclear.
A moment’s pause: putative nucleosome-based influences on MeCP2 regulationThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.
Authors: Thambirajah Anita A. , Ausió Juan
Published: November 2009

There has been a hotbed of activity surrounding MeCP2 research in the past number of years. Despite better characterizing the functions and nature of this protein, it has become abundantly clear that MeCP2 is involved in far more complex activities than perhaps initially anticipated. Recent publications have shown that MeCP2 is dynamically post-translationally modified, and it is possible that these marks permit MeCP2 to inhabit very diverse chromatin environments. It is also of interest to consider how nucleosome composition differs in these varying chromatin regions, and how the chromatin template itself contributes to diversifying the regulatory roles of MeCP2. These will be critical points to examine when seeking to understand how MeCP2 behaviour differentiates in tissues other than the brain. By understanding the chromatin and (or) tissue context in which MeCP2 interacts, it may be possible to discern the specific etiology of diseases linked to MeCP2 dysfunction.
Polycomb group protein gene silencing, non-coding RNA, stem cells, and cancerThis paper is one of a selection of papers published in this Special Issue, entitled The 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal's usual peer review process.
Authors: Gieni Randall S. , Hendzel Michael J.
Published: November 2009

Epigenetic programming is an important facet of biology, controlling gene expression patterns and the choice between developmental pathways. The Polycomb group proteins (PcGs) silence gene expression, allowing cells to both acquire and maintain identity. PcG silencing is important for stemness, X chromosome inactivation (XCI), genomic imprinting, and the abnormally silenced genes in cancers. Stem and cancer cells commonly share gene expression patterns, regulatory mechanisms, and signalling pathways. Many microRNA species have oncogenic or tumor suppressor activity, and disruptions in these networks are common in cancer; however, long non-coding (nc)RNA species are also important. Many of these directly guide PcG deposition and gene silencing at the HOX locus, during XCI, and in examples of genomic imprinting. Since inappropriate HOX expression and loss of genomic imprinting are hallmarks of cancer, disruption of long ncRNA-mediated PcG silencing likely has a role in oncogenesis. Aberrant silencing of coding and non-coding loci is critical for both the genesis and progression of cancers. In addition, PcGs are commonly abnormally overexpressed years prior to cancer pathology, making early PcG targeted therapy an option to reverse tumor formation, someday replacing the blunt instrument of eradication in the cancer therapy arsenal.
NuA4 and SWR1-C: two chromatin-modifying complexes with overlapping functions and componentsThis paper is one of a selection of papers published in this Special Issue, entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal's usual peer review process.
Authors: Lu Phoebe Y.T. , Lévesque Nancy , Kobor Michael S.
Published: November 2009

Chromatin structure is important for the compaction of eukaryotic genomes, thus chromatin modifications play a fundamental role in regulating many cellular processes. The coordinated activities of various chromatin-remodelling and -modifying complexes are crucial in maintaining distinct chromatin neighbourhoods, which in turn ensure appropriate gene expression, as well as DNA replication, repair, and recombination. SWR1-C is an ATP-dependent histone deposition complex for the histone variant H2A.Z, whereas NuA4 is a histone acetyltransferase for histones H4, H2A, and H2A.Z. Together the NuA4 and SWR1-C chromatin-modifying complexes alter the chromatin structure through 3 distinct modifications in yeast: post-translational addition of chemical groups, ATP-dependent chromatin remodelling, and histone variant incorporation. These 2 multi-protein complexes share 4 subunits and function together to regulate the circuitry of H2A.Z biology. The components and functions of both multi-protein complexes are evolutionarily conserved and play important roles in multi-cellular development and cellular differentiation in higher eukaryotes. This review will summarize recent findings about NuA4 and SWR1-C and will focus on the connection between these complexes by investigating their physical and functional interactions through eukaryotic evolution.
Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter
Authors: Sun Huigang , Bie Xiaomei , Lu Fengxia , Lu Yaping , Wu Yundailai , Lu Zhaoxin
Published: November 2009

Bacillus subtilis fmbR-1 was obtained from wild-type B. subtilis fmbR by replacement of the native promoter of the surfactin operon with the inducible promoter Pspac. The recombinant B. subtilis fmbR-1 produced more antibacterial substances than the wild-type strain. The overproducing phenotype was related to the enhancement of antagonistic activities against Bacillus cereus . HPLC peaks of surfactin for recombinant fmbR-1 showed patterns of lipopeptides similar to those of the wild-type strain, and surfactin production of the recombinant fmbR-1 was up to about fivefold greater than that of the wild-type strain without induction by isopropyl β-d-1-thiogalactopyranoside. However, the production of surfactin increased to about 10-fold more than that of the wild-type strain when it was induced by isopropyl β-d-1-thiogalactopyranoside.
Evaluation of the production of paralytic shellfish poisoning toxins by extracellular bacteria isolated from the toxic dinoflagellateAlexandrium minutum
Authors: Prol María Jesús , Guisande Cástor , Barreiro Aldo , Míguez Beatriz , de la Iglesia Pablo , Villar Adriano , Gago-Martínez Ana , Combarro María Pilar
Published: November 2009

The purpose of the study was to determine if paralytic shellfish poisoning (PSP) toxins are present in extracellular bacteria isolated from a toxic strain of the dinoflagellate Alexandrium minutum . A quantitative analysis was carried out of viable culturable bacteria attached to the surface of dinoflagellates and of bacteria present in dinoflagellate culture medium. A numerical taxonomy study was undertaken for presumptive identification of bacteria attached to the surface of dinoflagellates. Members of the following genera were detected on the cell surface of A. minutum: Cellulophaga , Marinomonas , Pseudoalteromonas , and Vibrio . The presence of intracellular PSP toxins in bacteria isolated from the cell surface of dinoflagellates was analysed by high-performance liquid chromatography with fluorescence detection (HPLC–FLD). Compounds that eluted at the same time as the standards of the PSP toxins GTX-2, GTX-3, GTX-4, dcGTX-2, and dcGTX-3 were present in some of the bacterial cell extracts. Natural fluorescent bacterial compounds, coeluting with some PSP toxins, were also detected. The results obtained showed that the fluorescent compounds, identified as putative PSP toxins by HPLC–FLD, did not correspond to any PSP analogue. This allowed us to reject the hypothesis that extracellular bacteria attached to the surface of dinoflagellates produce PSP toxins.
Cells in shearable and nonshearable regions ofSalmonella entericaserovar Enteritidis biofilms are morphologically and physiologically distinct
Authors: Mangalappalli-Illathu Anil K. , Lawrence John R. , Korber Darren R.
Published: November 2009

Cellular morphology, exopolymer chemistry, and protein expression of shearable and nonshearable fractions of Salmonella enterica serovar Enteritidis biofilms were examined. Biofilms were grown at a laminar flow velocity of 0.07 cm·s–1for ~120 h, resulting in biofilms with a thickness (mean ± SD) of 43 ± 24 µm. An empirically determined shear-inducing flow (1.33 cm·s–1) was then applied for 5 min, effectively reducing biofilm thickness by ~70% and leaving 13 ± 6 µm of nonshearable material and allowing fractionation of biofilm material into shearable and nonshearable regions. In situ lectin binding analyses revealed that there was no significant difference in the exopolymer glycoconjugate composition of the shearable and nonshearable biofilm zones. Length to width indices of cells from nonshearable and shearable biofilm regions as well as planktonic cells from biofilm effluent and continuous culture were determined to be 3.2, 2.3, 2.2, and 1.7, respectively, indicating that the cells in the shearable fraction were morphologically more similar to planktonic cells than the cells in the nonshearable biofilm fraction. Enhanced expression of proteins involved in cold shock response, adaptation, and broad regulatory functions (CspA, GrcA, and Hns, respectively) in cells from the shearable region as well as protein translation and modification and enhanced expression of protein involved in heat shock response and chaperonin function (DnaK) in cells from the nonshearable region revealed that the physiological status of cells in the two biofilm regions was distinct. This was also reflected in the different morphologies of cells from the two biofilm zones. Stratified patterns of cell metabolism and morphology in biofilms, obtained using shear-induced biofilm fractionation, may yield important information of how cells of deeply embedded biofilm bacteria survive imposed conditions of stress such as treatment with antimicrobial agents or antibiotics.
Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24
Authors: Duan Gaofei , Han Feng , Yu Wengong
Published: November 2009

The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 °C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0–10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+ can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of ~30 kDa, was found in AlyPI, which belongs to the ~60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.
TheBCG1619cgene is not essential for invasion and intracellular persistence ofMycobacterium bovisBCG in human THP-1 and A549 cell lines
Authors: Florio Walter , Brancatisano Franca Lisa , Bottai Daria , Esin Semih , Di Luca Mariagrazia , Counoupas Claudio , Maisetta Giuseppantonio , Lupetti Antonella , Batoni Giovanna , Campa Mario
Published: November 2009

The BCG1619c gene of Mycobacterium bovis bacillus Calmette–Guérin (BCG) encodes for a 24 kDa invasin-like protein and is identical to the Rv1566c gene of Mycobacterium tuberculosis . To assess whether this protein was necessary for entry and (or) intracellular persistence in professional phagocytes and (or) in lung epithelial cells, a BCG1619c knockout mutant of M. bovis BCG was generated and compared with the parental BCG strain for its ability to infect and multiply in human monocyte derived THP-1 cells and in the lung epithelial cell line A549. No significant difference between the mutated and the parental BCG strain was observed in either of these in vitro infection systems, indicating that the BCG1619c gene is not essential for cell invasion and intracellular growth of BCG.
Comparison of commercial viral genomic extraction kits for the molecular detection of foodborne viruses
Authors: Brassard Julie , Lamoureux Lisyanne , Gagné Marie-Josée , Poitras Élyse , Trottier Yvon-Louis , Houde Alain
Published: November 2009

When genetic material is extracted from viruses responsible for food illnesses, two broad types of possibilities are offered: conventional methods, which are well established but usually long and exacting to perform, or commercial kits, which are faster and easy to use but much more expensive. Thus, it is important to evaluate some performance parameters such as the analytical sensitivity to be able to select the optimal technique for each situation. The principal objective of this study was to establish and compare the analytical sensitivities of three commercial genetic material extraction methods (TRIzol reagent, FTA cards, and QIAGEN kits) along with three selected viruses, adenovirus, hepatitis A virus, and rotavirus. Viral detection was carried out using a standard PCR technique for adenovirus and reverse transcription PCR for rotavirus and hepatitis A virus. The results obtained showed that with the QIAGEN kit, the sensitivity was 2 logs lower than with the two other methods for all three viruses studied. Nevertheless, despite their lower analytical sensitivities, the other two extraction methods should not be overlooked and ought to be considered when evaluating the most efficient approach suitable for a specific commodity, since food-related outbreaks may be traced to a wide variety of food types.
Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose
Authors: Lopes Mateus Schreiner Garcez , Gomez José Gregório Cabrera , Silva Luiziana Ferreira
Published: November 2009

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101. The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa . It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl– mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.
Fibrinolytic enzymes from a newly isolated marine bacteriumBacillus subtilisA26: characterization and statistical media optimization
Authors: Agrebi Rym , Haddar Anissa , Hajji Mohamed , Frikha Fakher , Manni Laila , Jellouli Kemel , Nasri Moncef
Published: November 2009

A fibrinolytic enzyme producing bacterium was isolated and identified as Bacillus subtilis A26 on the basis of the 16S rRNA gene sequence. The fibrin zymography analysis reveals the presence of at least three fibrinolytic enzymes. The crude enzyme exhibited maximal activity at 60 °C and pH 8.0. Medium composition and culture conditions for the enzyme production by B. subtilis A26 were optimized using two statistical methods. The Plackett–Burman statistical design was applied to find the key ingredients and conditions for the best yield of enzyme production. Five significant variables (hulled grain of wheat, casein peptone, NaCl, CaCl2, and initial pH) were selected for the optimization studies. The response surface methodological approach was used to determine the optimal concentrations and conditions. The optimized medium contained 40.0 g·L–1hulled grain of wheat, 3.53 g·L–1casein peptone, 4.0 g·L–1CaCl2, 3.99 g·L–1NaCl, 0.01 g·L–1MgSO4, and 0.01 g·L–1KH2PO4, pH 7.78. The medium optimization resulted in a 4.2-fold increased level of fibrinolytic production (269.36 U·mL–1) compared with that obtained with the initial medium (63.45 U·mL–1). A successful and significant improvement in the production of protease by the A26 strain was accomplished using inexpensive carbon substrate (hulled grain of wheat), allowing a significant reduction in the cost of medium constituents.
The effects of tea polyphenols onCandida albicans: inhibition of biofilm formation and proteasome inactivation
Authors: Evensen Nikki A. , Braun Phyllis C.
Published: November 2009

The adherence of Candida albicans to one another and to various host and biomaterial surfaces is an important prerequisite for the colonization and pathogenesis of this organism. Cells in established biofilms exhibit different phenotypic traits and are inordinately resistant to antimicrobial agents. Recent studies have shown that black and green tea polyphenols exhibit both antimicrobial and strong cancer-preventive properties. Experiments were conducted to determine the effects of these polyphenols on C. albicans. Standard growth curves demonstrated a 40% reduction in the growth rate constant (K) with a 2 mg/mL concentration of Polyphenon 60, a green tea extract containing a mixture of polyphenolic compounds. Cultures treated with 1.0 µmol/L –(–)epigallocatechin-3-gallate (EGCG), the most abundant polyphenol, displayed a 75% reduction of viable cells during biofilm formation. Established biofilms treated with EGCG were also reduced, by 80%, as determined through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) colorimetric assays. Identical concentrations of epigallocatechin and epicatechin-3-gallate demonstrated similar biofilm inhibition. Further investigations regarding the possible mechanism of polyphenol action indicate that in vivo proteasome activity was significantly decreased when catechin-treated yeast cells were incubated with a fluorogenic peptide substrate that measured proteasomal chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities. Impairment of proteasomal activity by tea polyphenols contributes to cellular metabolic and structural disruptions that expedite the inhibition of biofilm formation and maintenance by C. albicans.
Molecular and morphological markers for rapid distinction between 2 Colletotrichum species
Authors: Pileggi Sônia Alvim Veiga , Vieira de Oliveira Sarah Franco , Andrade Caroline Waculicz , Vicente Vânia Aparecida , Dalzoto Patrícia do Rocio , Kniphoff da Cruz Gerson , Gabardo Juarez , Massola Nelson Sidnei , Tozze Hugo José , Pileggi Marcos , Kava-Cordeiro Vanessa , Galli-Terasawa Lygia Vitória , Pimentel Ida Chapaval , Stringari Danyelle , Glienke Chirlei
Published: November 2009

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella ) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (“espinheira-santa”) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense . Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Col1 and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.
A refrigeration temperature of 4 °C does not prevent static growth of Yersinia pestis in heart infusion broth
Authors: Torosian Stephen D. , Regan Patrick M. , Doran Tara , Taylor Michael A. , Margolin Aaron
Published: November 2009

Multiple barriers such as inspections, testing, and proper storage conditions are used to minimize the risk of contaminated food. Knowledge of which barriers, such as refrigeration, are effective in preventing pathogen growth and persistence, can help direct the focus of efforts during food sampling. In this study, the doubling times were evaluated for 10 strains of Yersinia pestis of different genetic background cultured in heart infusion broth (HIB) kept at 4 °C ± 1 °C under static conditions. Nine out of the 10 strains were able to grow at 4 °C ± 1 °C. Apparent doubling times for 7 of the strains ranged from 41 to 50 h. Strain Harbin and strain D1 had apparent doubling times of 65 and 35 h, respectively, and strain O19 Ca–6 did not grow at all. Analysis of variance showed that the averaged growth data (colony forming units per mL) between strains that grew were not significantly different. The data presented here demonstrate that refrigeration alone is not an effective barrier to prevent static growth of Y. pestis in HIB. These findings provide the preliminary impetus to investigate Y. pestis growth in a variety of food matrices that may provide a similar environment as HIB.
Detection of Yersinia pestis over time in seeded bottled water samples by cultivation on heart infusion agar
Authors: Torosian Stephen D. , Regan Patrick M. , Taylor Michael A. , Margolin Aaron
Published: November 2009

The viable persistence of Yersinia pestis seeded in bottled spring water was evaluated by performing 2 studies that involved inoculating a total of 21 different test strains into individual 500 mL reservoirs. Approximately 2 × 104 CFU/mL of Y. pestis was inoculated into each reservoir and held for sampling at 26 °C ± 1 °C. In study No. 2, 9 strains (Harbin, Nepal, UNH 1A, UNH 1B, ZE94, CO92, PB6, PB6 DP, and Pexu) could no longer be recovered using a plate count assay between 79 and 138 days post-seeding; other strains (K25 lcr, O19 Ca–6, and K25 pst) could no longer be recovered between 112 and 160 days post seeding. The data generated in this study demonstrate that certain strains of Y. pestis can remain viable in bottled water for extended periods of time.
The role ofAcidithiobacillus ferrooxidansin alleviating the inhibitory effect of thiosulfate on the growth of acidophilicAcidiphiliumspecies isolated from acid mine drainage samples from Garubathan, India
Authors: Gurung Anirudra , Chakraborty Ranadhir
Published: November 2009

Several acidophilic chemolithoautotrophic and heterotrophic strains were isolated from acid mine drainage samples from Garubathan, West Bengal, India. The strains, chemolithoautotrophic DK6.1 and heterotrophic DKAP1.1, used in this study were assigned to the species Acidithiobacillus ferrooxidans and Acidiphilium cryptum , respectively. Unamended filtered and subsequently autoclaved elemental sulfur spent medium of A. ferrooxidans was used as the medium to study heterotrophic growth of A. cryptum DKAP1.1. While characterizing the heterotrophic strain, an inhibitory effect of thiosulfate on A. cryptum DKAP1.1 was identified. The lethality of thiosulfate broth was directly related to the concentration of thiosulfate in the medium. Nonviability of A. cryptum DKAP1.1 in the presence of thiosulfate was alleviated by A. ferrooxidans DK6.1 in co-culture. Microbiological data on a positive growth effect for A. ferrooxidans DK6.1 caused by co-culturing in solid media in the presence of A. cryptum DKAP1.1 is also presented.
The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation
Authors: Tang Wei , Zhang Sufang , Wang Qian , Tan Haidong , Zhao Zongbao Kent
Published: November 2009

The oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids to over 60% of its cell dry mass under a nitrogen-limited condition. We showed that extracellular and intracellular citrate concentrations of L. starkeyi AS 2.1560 increased and the nicotinamide adenine dinucleotide – isocitrate dehydrogenase (NAD+–IDH) activity decreased at the beginning of the lipid accumulation, suggesting that the attenuation of the NAD+–IDH activity might initiate lipid storage. We next cloned the IDH gene by the methods of degenerate PCR and rapid amplification of cDNA ends. Phylogenetic analyses of the evolutionary relationships among LsIDH1, LsIDH2, and other yeast NAD+–IDHs revealed that the L. starkeyi IDH had a closer relationship with the IDHs of Yarrowia lipolytica . Further real-time PCR analysis showed that the expression levels of both LsIDH1 and LsIDH2 decreased concurrently with the evolution of cellular lipids. Our data should be valuable for understanding the biology of oleaginous yeasts and for further strain engineering of L. starkeyi.
Strain construction for enhanced production of spinosad via intergeneric protoplast fusion
Authors: Wang Chao , Zhang XiaoLin , Chen Zhi , Wen Ying , Song Yuan
Published: November 2009

Spinosad is a new class of insecticides produced by Saccharopolyspora spinosa . The aim of this study was to construct a starch-utilizing strain that overproduced spinosad by intergeneric fusion between S. spinosa and Streptomyces avermitilis . Protoplast fusion is an important technique for engineering microbial strains, especially for microorganisms with few available molecular genetic tools. Protoplast fusion was conducted with UV-irradiated protoplasts of S. spinosa and S. avermitilis. Among 76 recombinants screened by ESI-MS and HPLC, a starch-utilizing strain F17, identified as S. spinosa, was obtained. The yield of spinosad in F17 was increased by 447.22%, compared with the yield of the wild-type strain. This is the first report of intergeneric protoplast fusion between S. spinosa and S. avermilitis, which shows great potential for industrial applications.
Compensatory periplasmic nitrate reductase activity supports anaerobic growth ofPseudomonas aeruginosaPAO1 in the absence of membrane nitrate reductase
Authors: Van Alst Nadine E. , Sherrill Lani A. , Iglewski Barbara H. , Haidaris Constantine G.
Published: November 2009

Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa . Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.
Optimization of medium and cultivation conditions forl-amino acid oxidase production byAspergillus fumigatus
Authors: Singh Susmita , Gogoi B. K. , Bezbaruah R. L.
Published: November 2009

A fungal strain was selected from the microbial repository of the North-East Institute of Science and Technology, Jorhat, India, which could produce a high yield of l-amino acid oxidase. 18SrRNA, ITS1, 5.8SrRNA ITS2, and partial 28 S rRNA sequencing and phenotypic characteristics indicate that it belong to the species Aspergillus fumigatus (designated as P13). Maximum production of enzyme (59.55 × 10−3 U/mg dry cell mass) was obtained in a medium containing 10 g/L glucose, 4 g/L yeast extract, and 4 g/L ammonium sulfate, with 20 mmol/L of l-threonine as the inducer. The optimum temperature for enzyme production was 30 °C at pH 7.0, with a shaking speed of 200 r/min. At 96 h, the enzyme activity was maximum. The A. fumigatus P13 l-amino acid oxidase accepts a broad substrate range, and the maximum enzyme activity (20.41 × 10−3 U/mg dry cell mass) was obtained with 50 mmol/L of dl-tyrosine. In the literature, no reports have been found regarding the production of l-amino acid oxidase by A. fumigatus. The enzyme showed enantiomerically pure amino acid formation, which has tremendous demand in industrial applications.
Phylogenetic analysis of methanotrophic communities in cover soils of a landfill in Ontario
Authors: Lin Bin , Monreal Carlos M. , Tambong James T. , Miguez Carlos B. , Carrasco-Medina Lorna
Published: November 2009

We examined the methanotrophs in the Trail Road Landfill soils, Ottawa, Ontario, through cultivation-independent molecular assay and the culturing approach. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified methanotroph-specific 16S rDNA gene fragments revealed a more diverse type I (RuMP pathway) methanotrophic community than type II (serine pathway) in 17 soil samples taken along a 50 m transect. The type II methanotrophic community was less diverse, with the dominance of Methylocystis in almost all samples, and clustering with high similarity (85%–88%). Also, the results showed that the C/N ratio of soil organic matter could significantly affect the methanotrophic community structures. The DGGE results were supported by sequence analysis of cloned pmoA. Members of the genera Methylobacter (type I), Methylocaldum (type X), and Methylocystis (type II) appeared to be the dominant methanotrophs. From methanotrophic enrichments, we isolated type I Methylobacter sp., and 3 type II Methylocystis spp.,which appeared to be one of the dominant bacteria species in the soil sample from which isolates were obtained.
Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil
Authors: Providenti Miguel A. , Begin Melissa , Hynes Samielle , Lamarche Christine , Chitty David , Hahn Jessica , Beaudette Lee A. , Scroggins Rick , Smith Myron L.
Published: November 2009

In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus , and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the ~1 × 109 to ~1 × 104 colony forming units (CFU)/g soil range. The detection limit was improved to ~1 × 102 CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilis strains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 × 102 CFU/g detection limit by days 28–105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.
GeneSMb21071of plasmid pSymB is required for osmoadaptation ofSinorhizobium meliloti1021 and is implicated in modifications of cell surface polysaccharides structure in response to hyperosmotic stress
Authors: Reguera María , Lloret Javier , Margaret Isabel , Vinardell José M. , Martín Marta , Buendía Ana , Rivilla Rafael , Ruiz-Sainz José E. , Bonilla Ildefonso , Bolaños Luis
Published: November 2009

Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti , implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that apparently encode surface polysaccharides. However, only 2 of these clusters, containing the exo and exp genes, have been associated with the synthesis of the acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The functions of the other 9 clusters remain unsolved. The involvement of one of those regions, pSymB cluster 3, on surface polysaccharide synthesis and its possible implication in osmoadaptation were investigated. In silico analysis of cluster 3 showed that it putatively encodes for the synthesis and transport of a methylated surface polysaccharide. Mutants affected in this cluster were symbiotically effective but showed defects in growth under saline and nonsaline osmotic stress. The gene SMb21071, encoding a putative initiating glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and silver staining showed that osmotic stresses changed the profiles of surface polysaccharides of wild-type and mutants strains in different ways. The overall results suggest that cluster 3 is important for growth under saline stress and essential for growth under nonsaline hyperosmotic stress, and it appears to be implicated in maintaining and (or) modifying surface polysaccharides in response to osmotic stress.
Phenotypic, genotypic, and symbiotic diversities in strains nodulating clover in different soils in Spain
Authors: Ramírez-Bahena Martha-Helena , Velázquez Encarna , Fernández-Santos Félix , Peix Alvaro , Martínez-Molina Eustoquio , Mateos Pedro F.
Published: November 2009

Trifolium species are the most common legumes present in wild Spanish soils; however, there are no studies to date on the diversity of rhizobia nodulating clover in Spain. Twenty strains from different Spanish soils with acidic, neutral, and basic pH were selected to study their genotypic, phenotypic, and symbiotic features. The results showed that the isolates were genotypically diverse, displaying 12 different DNA fingerprint patterns and also 14 different plasmid profiles. Although they have 16S rRNA gene sequences that are nearly identical to that of the type strain of Rhizobium leguminosarum , their recA and atpD gene sequences were phylogenetically divergent from those of R. leguminosarum reference strains, and phenotypic divergence as well as different host ranges were also found. Although most of them nodulated both Trifolium and Phaseolus , only 5 strains were also able to nodulate Pisum . The results of the effectiveness analysis showed a high variability in the symbiotic characteristics of our strains and suggested that Pisum is the more restrictive host of this group. Interestingly, some of the Trifolium isolates showed an ability to promote growth of Pisum in the absence of nodulation.
Planktonic cell yield is linked to biofilm development
Authors: Bester Elanna , Edwards Elizabeth A. , Wolfaardt Gideon M.
Published: November 2009

We report on the ability of surface-associated microbes to produce and release single planktonic cells to the bulk liquid as early as 6 h after attachment, with pure culture and mixed-species biofilms yielding up to ~1 × 106 cells/cm2 of attachment area per hour to the effluent after 24 h. Planktonic cell production typically increased as the biofilm developed and levelled off after the biofilm reached steady-state dimensions. Microscopic observations of continuous-flow cultured biofilms revealed independent cell movement within the biofilm microenvironment compared with flow-dependent movement of mostly single cells in the bulk-liquid phase. These results indicate that the prevailing concept of detachment occurring only after the biofilm has matured is incomplete. Instead, we show that biofilms yield cells to the environment soon after initial surface contact; the extent of this yield is dependent on biofilm development, which in turn is influenced by environmental parameters such as bulk-liquid flow rates and nutrient availability. The observation that biofilms yield significant numbers of cells throughout development should lead to a greater understanding of pathogen dissemination, biofouling of products or facilities, and the role that biofilms play in microbial proliferation in the environment.
Strain competition and agar affect the interaction of rhizobia with rice
Authors: Perrine-Walker Francine M. , Hynes Michael F. , Rolfe Barry G. , Hocart Charles H.
Published: November 2009

Competition assays with Sinorhizobium meliloti 1021 and its GFP-labelled pSymA cured and deleted derivatives, SmA818 and SmA146, demonstrated that Sm1021 could still inhibit rice seedling growth even when outnumbered by a large excess of the noninhibitory cured or deleted strain. The wild-type strain Sm1021 also inhibited the growth of its noninhibitory pSymA-cured strain SmA818(gfp) and its pSymA-deleted strain SmA146(gfp) in a manner suggesting that Sm1021 produced a bacteriocin-like substance. The production of, and resistance to, this substance seemed to be pSymA-associated, but it was not the cause of killing in competition experiments on rice, suggesting that the killing of SmA818(gfp) and SmA146(gfp) was medium dependent. The addition of agar in liquid F10 medium at concentrations ≤0.4% (m/v) abolished the rice growth inhibition of strain Sm1021 and Sm1021(gfp). The increased medium viscosity at higher agar concentrations decreased the diffusion of gases and small molecules through the media. Thus, the low agar concentrations may mimic waterlogged soil conditions leading to the production of inhibitory compounds by the bacterial strains under microaerobic conditions.
Mitochondrial oxidative phosphorylation in hearts subjected to Ca2+ depletion and Ca2+ repletionThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.
Authors: Makazan Zhanna , Saini-Chohan Harjot K. , Dhalla Naranjan S.
Published: November 2009

Repletion of Ca2+ in the Ca2+-depleted heart has been shown to produce cardiac dysfunction, myocardial cell damage, intracellular Ca2+ overload, and defects in sarcolemmal and sarcoplasmic reticulum function (Ca2+ paradox). Although these alterations in the Ca2+-paradox heart are associated with a depression in the high-energy phosphate stores, little information regarding changes in mitochondrial oxidative phosphorylation is available. Perfusion of rat hearts with Ca2+-free medium for 5 min followed by reperfusion with a medium containing 1.25 mmol/L Ca2+ for 10 min depressed mitochondrial state 3 respiration, respiratory control index, ADP/O ratio, and rate of oxidative phosphorylation without any change in state 4 respiration. These alterations were partially prevented when the reperfusion was carried out with a medium containing low Ca2+ (0.10–0.50 mmol/L). Treatment of heart with inhibitors of sarcolemmal Ca2+ channels (verapamil and diltiazem) or inhibitors of Na+/Ca2+ exchange (KB-R7943) and Na+/H+ exchange (amiloride) failed to modify changes in mitochondrial function due to Ca2+ paradox. Likewise, antioxidants N-acetylcysteine and N-(2-mercaptopropionyl)-glycine and an oxyradical-scavenging mixture of superoxide dismutase and catalase were ineffective in preventing the mitochondrial alterations in the Ca2+-paradox heart. Incubation of mitochondria with various concentrations of Ca2+ inhibited oxidative phosphorylation; this Ca2+-induced change in mitochondrial function was not affected by different oxyradical-scavenging systems. These observations suggest that defects in mitochondrial function in the Ca2+-paradox heart may be due to the occurrence of intracellular Ca2+ overload rather than the development of oxidative stress.
IGF-1 receptor transactivation mediates Src-dependent cortactin phosphorylation in response to angiotensin IIThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.
Authors: Zahradka Peter , Storie Benjamin , Wright Brenda
Published: November 2009

Release of angiotensin II (Ang II) after vascular injury promotes tissue repair by stimulating phenotypic modulation of smooth muscle cells, which enables cell proliferation and migration. This process requires cytoskeleton remodeling, which involves cortactin, a scaffold protein that is phosphorylated by Src kinase in response to Ang II. Since insulin-like growth factor (IGF)-1 receptor transactivation mediates intracellular signals originating from the Ang II type 1 (AT1) receptor in a Src kinase-dependent manner, we examined whether IGF-1 receptor transactivation was also required for cortactin phosphorylation. Treatment of quiescent smooth muscle cells with Ang II resulted in both cortactin phosphorylation and its translocation to the plasma membrane. Both events were prevented by 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo(3,4-d)pyrimidin-4-amine (PP1), a Src kinase inhibitor, and by AG1024, an inhibitor of the IGF-1 receptor tyrosine kinase. Additionally, PP1 and AG1024 blocked the association of cortactin with actin-related protein (Arp) 3, an actin nucleation factor. These results indicate that Src kinase and the IGF-1 receptor kinase are necessary for activating cortactin. Phosphorylation of Src kinase in Ang II-treated cells was subsequently examined and was shown to be prevented by AG1024. Furthermore, Src kinase phosphorylation was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphatidylinositol (PI) 3-kinase. These data establish that IGF-1 receptor transactivation is required for Src kinase-mediated cortactin phosphorylation and cytoskeletal reorganization in response to Ang II.
S100A8/A9: a mediator of severe asthma pathogenesis and morbidity?This article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.
Authors: Halayko Andrew J. , Ghavami Saeid
Published: November 2009

Nearly 12% of children and 6% of adults in Canada have been diagnosed with asthma. Although in most patients symptoms are controlled by inhaled steroids, a subpopulation (∼10%) characterized by excessive airway neutrophilia, is refractory to treatment; these patients exhibit severe disease, and account for more than 50% of asthma health care costs. These numbers underscore the need to better understand the biology of severe asthma and identify pro-asthma mediators released by cells, such as neutrophils, that are unresponsive to common steroid therapy. This review focuses on a unique protein complex consisting of S100A8 and S100A9. These subunits belong to the large Ca2+-binding S100 protein family and are some of the most abundant proteins in neutrophils and macrophages. S100A8/A9 is a damage-associated molecular pattern (DAMP) protein complex released in abundance in rheumatoid arthritis, inflammatory bowel disease, and cancer, but there are no definitive studies on its role in inflammation and obstructive airways disease. Two receptors for S100A8/A9, the multiligand receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4), are expressed in lung. TLR4 is linked with innate immunity that programs local airway inflammation, and RAGE participates in mediating fibroproliferative remodeling in idiopathic pulmonary fibrosis. S100A8/A9 can induce cell proliferation, or apoptosis, inflammation, collagen synthesis, and cell migration. We hypothesize that this capacity suggests S100A8/A9 could underpin chronic airway inflammation and airway remodeling in asthma by inducing effector responses of resident and infiltrating airway cells. This review highlights some key issues related to this hypothesis and provides a template for future research.