bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Aspergillus fumigatus conidia binding to extracellular matrix proteins by sialic acids: a pH effect?
Authors: Tiralongo Joe , Wohlschlager Therese , Tiralongo Evelin , Kiefel Milton J.
Published: August 2009

Infection by Aspergillus fumigatus, which causes the life-threatening disease invasive aspergillosis, begins with the inhalation of conidia that adhere to and germinate in the lung. Previous studies have shown that A. fumigatus conidia express high levels of the negatively charged 9-carbon sugar sialic acid, and that sialic acid appears to mediate the binding of A. fumigatus conidia to basal lamina proteins. However, despite the ability of sialic acid to inhibit adherence of A. fumigatus conidia, the exact mechanism by which this binding occurs remains unresolved. Utilizing various free sialic acids and other carbohydrates, sialic acid derivatives, sialoglycoconjugates, glycoproteins, α-keto acid related compounds and amino acids we have found that the binding of A. fumigatus conidia to type IV collagen and fibrinogen was inhibited by (i) glycoproteins (in a sialic acid-independent manner), and (ii) free sialic acids, glucuronic acid and α-keto acid related compounds. However, inhibition by the latter was found to be the result of a shift in pH from neutral (pH 7.4) to acidic (less than pH 4.6) induced by the relatively high concentrations of free sialic acids, glucuronic acid and α-keto acid related compounds used in the binding assays. This suggests that previous reports describing inhibition of A. fumigatus conidia binding by free sialic acid may actually be due to a pH shift similar to that shown here. As previously reported, we found that A. fumigatus conidia express only N-acetylneuraminic acid, the most common sialic acid found in nature. However, A. fumigatus appears to do so by an alternative mechanism to that seen in other organisms. We report here that A. fumigatus (i) does not incorporate sialic acid obtained from the environment, (ii) does not synthesize and incorporate sialic acid from exogenous N-acetylmannosamine, and (iii) lacks homologues of known sialic acid biosynthesizing enzymes.
Expression of the Streptococcus mutans essential two-component regulatory system VicRK is pH and growth-phase dependent and controlled by the LiaFSR three-component regulatory system
Authors: Tremblay Yannick D. N. , Lo Henry , Li Yung-Hua , Halperin Scott A. , Lee Song F.
Published: August 2009

As an inhabitant of the human oral cavity, Streptococcus mutans faces frequent environmental changes. Two-component regulatory systems (TCSs) play a critical role in responding to these changes. Recently, an essential TCS, VicRKX, has been identified. The objective of this study was to identify the environmental signal and bacterial factors regulating the expression of the vicRKX operon. The promoter of the vicRKX operon was fused to a promoterless lacZ reporter gene and introduced into S. mutans UA159. LacZ plate assay identified pH, vancomycin, ampicillin, penicillin G and polymyxin B, but not carbohydrates, as factors affecting expression. Using RNA dot-blotting, high levels of vicR transcript were observed in cells at the mid- and late-exponential phase of growth and in cells grown in media buffered at pH 7.8. Given that vicR expression was pH-dependent, the genes encoding a putative pH-sensing three-component regulatory system (LiaFSR) were deleted. The liaS mutant exhibited upregulation of vicR regardless of the growth condition. The role of VicK, VicX, and the competence-signal peptide (CSP) was also investigated; the results showed that vicR expression was not autoregulated and was downregulated by the CSP in a ComX-independent manner. In conclusion, the expression of vicRKX is influenced by culture pH, growth phase and antibiotic stress, and is regulated by LiaFRS.
Identification and characterization of target genes of the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius
Authors: Iida Aya , Ohnishi Yasuo , Horinouchi Sueharu
Published: August 2009

The GinI/GinR quorum-sensing system represses oxidative fermentation, including acetic acid and gluconic acid fermentation, as well as antifoam activity in Gluconacetobacter intermedius NCI1051. An 89 aa protein, GinA, whose production is induced by the quorum-sensing system, represses both oxidative fermentation and antifoam activity via a still unknown mechanism, although an OmpA family protein, GmpA, as a target of the GinI/GinR quorum-sensing system via GinA, has been found to repress oxidative fermentation. In this study, four novel GinA-inducible genes (gltA, pdeA, pdeB and nagA) were identified and their involvement in oxidative fermentation and antifoam activity was examined by gene disruption. Disruption of nagA (which encodes a putative N-acetylglucosamine-6-phosphate deacetylase) decreased the growth rate in the exponential growth phase, indicating that nagA was required for the rapid growth of the strain. This unexpected finding revealed a new aspect of the GinI/GinR quorum-sensing system: it accelerates exponential growth by induction of nagA. In contrast, gltA (a putative glycosyltransferase) and pdeA (a putative cyclic-di-GMP phosphodiesterase) were shown to repress oxidative fermentation, including acetic acid and gluconic acid fermentation. gltA was also shown to repress antifoam activity. Disruption of pdeB (a putative phosphodiesterase/diguanylate cyclase) caused no phenotypic changes. Taking our previous results into consideration, these results showed an apparently complex mechanism for repressing oxidative fermentation by the quorum-sensing system; at least three GinA-inducible genes, gltA, pdeA and gmpA, were involved in the repression of oxidative fermentation by the GinI/GinR quorum-sensing system, the most characteristic feature of the acetic acid bacteria.
Burkholderia cenocepacia zinc metalloproteases influence resistance to antimicrobial peptides
Authors: Kooi Cora , Sokol Pamela A.
Published: August 2009

Burkholderia cenocepacia secretes two zinc-dependent metalloproteases, designated ZmpA and ZmpB. Previously, ZmpA and ZmpB have been shown to cleave several proteins important in host defence. In this study, the ability of ZmpA and ZmpB to digest and inactivate antimicrobial peptides involved in innate immunity was examined. ZmpB but not ZmpA cleaved β-defensin-1. ZmpA but not ZmpB cleaved the cathelicidin LL-37. Both enzymes cleaved elafin and secretory leukocyte inhibitor, which are antimicrobial peptides as well as neutrophil elastase inhibitors. Both ZmpA and ZmpB cleaved protamine, a fish antimicrobial peptide, and a zmpA zmpB mutant was more sensitive to protamine killing than the parental strain. ZmpA or ZmpB cleavage of elafin inactivated its anti-protease activity. The effect of ZmpA and ZmpB on the neutrophil proteases elastase and cathepsin G was also examined but neither enzyme was active against these host proteases. These studies suggest that ZmpA and ZmpB may influence the resistance of B. cenocepacia to host antimicrobial peptides as well as alter the host protease/anti-protease balance in chronic respiratory infections.
Pet secretion, internalization and induction of cell death during infection of epithelial cells by enteroaggregative Escherichia coli
Authors: Betancourt-Sanchez Miguel , Navarro-Garcia Fernando
Published: August 2009

In an in vitro model using HEp-2 cells treated with purified plasmid-encoded toxin (Pet), we have identified morphological changes characterized by cell rounding and detachment after toxin internalization; these changes progress to cell death. However, these effects have not yet been shown to occur during the infection of epithelial cells by enteroaggregative Escherichia coli (EAEC). Here, we show that the secretion of Pet by EAEC is regulated at the transcriptional level, since secretion was inhibited in eukaryotic cell culture medium, although Pet was efficiently secreted in the same medium supplemented with tryptone. Inefficient secretion of Pet by EAEC in DMEM prevented cell detachment, whereas efficient Pet secretion in DMEM/tryptone increased cell detachment in a HEp-2 cell adherence assay. Interestingly, Pet toxin was efficiently delivered to epithelial cells, since it was internalized into epithelial cells infected with EAEC at similar concentrations to those obtained by using 37 μg ml−1 purified Pet protein. Additionally, Pet was not internalized when the epithelial cells were infected with a pet clone, HB101(pCEFN1), unlike the wild-type strain, which has a high adherence capability. There is a correlation between Pet secretion by EAEC, the internalization of Pet into epithelial cells, cell detachment and cell death in EAEC-infected cells. The ratio between live and dead cells decreased in cells treated with wild-type EAEC in comparison with cells treated with an isogenic mutant in the pet gene, whereas the effects were restored by complementing the mutant with the pet gene. All these data indicate that Pet is an important virulence factor in the pathogenesis of EAEC infection.
Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana
Authors: Wanchoo Arun , Lewis Michael W. , Keyhani Nemat O.
Published: August 2009

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host–pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.
Mode of vegetative reproduction of the bipolar budding yeast species Wickerhamomyces pijperi and related strains
Authors: Imanishi Yumi , Jindamorakot Sasitorn , Limtong Savitree , Nakase Takashi
Published: August 2009

To clarify the budding pattern of Wickerhamomyces pijperi, the vegetative cells were observed by scanning electron microscopy. The cells grew by bipolar budding, but cells that budded from the shoulder of a mother cell were occasionally observed. We examined the cell morphology and phylogeny of five strains of Wickerhamomyces sp. isolated in Thailand as well as seven W. pijperi and three Wickerhamomyces sp. strains that were preserved in culture collections. Phylogenetic analysis based on three different nucleotide sequences (D1/D2 domain of 26S rDNA, the actin gene ACT1 and the elongation factor 2 gene EF2) indicated that all the strains belonged to the genus Wickerhamomyces and were neighbours of the type strain W. pijperi NBRC 1290T. The strains fell into two groups in this analysis. The budding patterns of the strains were carefully observed by staining the bud scars, and these patterns were categorized into three groups: types I–III. Type I included cells that grew by bipolar budding and formed multiple scars, type III included cells that grew by multilateral budding and formed a single scar, and type II included cells that exhibited a mixture of type I and type III patterns. Among the 15 strains, 12 strains, including W. pijperi NBRC 1290T, mainly exhibited type I or type II budding patterns; these strains belonged to group 1 of the phylogenetic analysis. The remaining three strains, which belonged to group 2, exhibited either type II or type III patterns. Thus the phylogenetic relationship and budding patterns are related. Moreover, some cells also exhibited budding characteristics that were intermediate between bipolar and multilateral budding.
Low pH regulates the production of deoxynivalenol by Fusarium graminearum
Authors: Gardiner Donald M. , Osborne Sheree , Kazan Kemal , Manners John M.
Published: August 2009

Fusarium graminearum, which causes the globally important head blight disease of wheat, is responsible for the production of the harmful mycotoxin deoxynivalenol (DON) in infected grain. The production of DON by F. graminearum occurs at much higher levels during infection than during axenic growth, and it is therefore important to understand how DON production is regulated in the fungus. Recently, we have identified amines as potent inducers of in vitro DON production in F. graminearum. Although amines strongly induced expression of the key DON biosynthesis gene TRI5 and DON production to levels equivalent to those observed during infection, the timing of this induction suggested that other factors are also likely to be important for the regulation of DON biosynthesis. Here we demonstrate that low extracellular pH both promotes and is required for DON production in F. graminearum. A combination of low pH and amines results in significantly enhanced expression of the TRI5 gene and increased DON production during axenic growth. A better understanding of DON production in F. graminearum would have implications in developing future toxin management strategies.
Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis
Authors: Tachon Sybille , Michelon Damien , Chambellon Emilie , Cantonnet Monique , Mezange Christine , Henno Lucy , Cachon Rémy , Yvon Mireille
Published: August 2009

The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.
Streptomyces roseoverticillatus produces two different poly(amino acid)s: lariat-shaped γ-poly(l-glutamic acid) and ϵ-poly(l-lysine)
Authors: Nishikawa Masanobu , Kobayashi Kei
Published: August 2009

The poly(amino acid)s γ-poly(dl-glutamic acid) (gPGA) and ϵ-poly(l-lysine) (ePL) are known to be natural linear poly(amino acid)s secreted by Bacillus spp. and Streptomyces spp., respectively. In this study, a Streptomyces strain producing both ePL and gPGA was identified. Mass spectrometry and other analyses revealed that the gPGA is a mixture of oligomers consisting of 10–13 l-glutamic acid residues linked by isopeptide bonds. In contrast to the known Bacillus gPGA, the glutamic acid oligomers have a cyclodehydrated structure in each molecule. We previously reported that the ePL molecules secreted by the same Streptomyces strain disperse only slightly in an agar culture plate, as though they were larger molecules. This phenomenon is explicable by the observed polyion complex formation between the glutamic acid oligomers and ePLs. The glutamic acid oligomers control the ePL's dispersion, which would also affect the spatial distribution of the ePL's antimicrobial activity. Therefore, gene clustering or common use of the gene was presumed for biosynthesis of the two poly(amino acid)s. However, no gene for biosynthesis of the glutamic acid oligomer was found in the neighbouring region of that for ePL biosynthesis, and the glutamic acid oligomer was produced by a mutant in which the ePL biosynthetic gene was inactivated by gene disruption.
Transcriptional analysis of the response of Neurospora crassa to phytosphingosine reveals links to mitochondrial function
Authors: Videira Arnaldo , Kasuga Takao , Tian Chaoguang , Lemos Catarina , Castro Ana , Glass N. Louise
Published: August 2009

Treatment ofNeurospora crassacells with phytosphingosine (PHS) induces programmed cell death (PCD) by an unknown mechanism. To determine the relationship between PHS treatment and PCD, we determined changes in global gene expression levels inN. crassaduring a time-course of PHS treatment. Most genes having differential expression levels compared to untreated samples showed an increase in relative expression level upon PHS exposure. However, genes encoding mitochondrial proteins were highly enriched among ∼100 genes that showed a relative decrease in expression levels after PHS treatment, suggesting that repression of these genes might be related to the death-inducing effects of PHS. Since mutants in respiratory chain complex I are more resistant to both PHS and hydrogen peroxide (H2O2) than the wild-type strain, possibly related to the production of reactive oxygen species, we also compared gene expression profiles of a complex I mutant (nuo14) and wild-type in response to H2O2. Genes with higher expression levels in the mutant, in the presence of H2O2, are also significantly enriched in genes encoding mitochondrial proteins. These data suggest that complex I mutants cope better with drug-induced decrease in expression of genes encoding mitochondrial proteins and may explain their increased resistance to both PHS and H2O2. As a way of identifying new components required for PHS-induced death, we analysed the PHS sensitivity of 24 strains carrying deletions in genes that showed a significant alteration in expression pattern when the wild-type was exposed to the sphingolipid. Two additional mutants showing increased resistance to PHS were identified and both encode predicted mitochondrial proteins, further supporting the role of the mitochondria in PHS-induced PCD.
The modular architecture of meningococcal factor H-binding protein
Authors: Beernink Peter T. , Granoff Dan M.
Published: August 2009

Meningococcal factor H binding protein (fHbp) is a promising vaccine antigen that binds the human complement downregulatory molecule factor H (fH), and this binding enhances the survival of the organism in serum. Based on sequence variability of the entire protein, fHbp has been divided into three variant groups or two subfamilies. Here, we present evidence based on phylogenetic analysis of 70 unique fHbp amino acid sequences that the molecular architecture is modular. From sequences of natural chimeras we identified blocks of two to five invariant residues that flanked five modular variable segments. Although overall, 46 % of the fHbp amino acids were invariant, based on a crystal structure, the invariant blocks that flanked the modular variable segments clustered on the membrane surface containing the amino-terminal lipid anchor, while the remaining invariant residues were located throughout the protein. Each of the five modular variable segments could be classified into one of two types, designated α or β, based on homology with segments encoded by variant 1 or 3 fHbp genes, respectively. Forty of the fHbps (57 %) comprised only α (n=33) or β (n=7) type segments. The remaining 30 proteins (43 %) were chimeras and could be classified into one of four modular groups. These included all 15 proteins assigned to the previously described variant 2 in subfamily A. The modular segments of one chimeric modular group had 96 % amino acid identity with those of fHbp orthologs in Neisseria gonorrhoeae. Collectively, the data suggest that recombination between Neisseria meningitidis and N. gonorrhoeae progenitors generated a family of modular, antigenically diverse meningococcal fHbps.
Class II one-peptide bacteriocins target a phylogenetically defined subgroup of mannose phosphotransferase systems on sensitive cells
Authors: Kjos Morten , Nes Ingolf F. , Diep Dzung B.
Published: August 2009

Membrane-located proteins (IIC and IID) of the mannose-phosphotransferase system (man-PTS) have previously been shown to serve as target receptors for several bacteriocins. Although many bacteria contain at least one such man-PTS in their genome, most bacteriocins display a narrow inhibitory spectrum, targeting predominantly bacteria closely related to the producers. In the present study we have analysed the receptor spectrum of one-peptide bacteriocins of class II. A phylogenetic analysis of 86 man-PTSs from a wide range of bacterial genera grouped the man-PTSs into three main clusters (groups I–III). Fourteen man-PTSs distributed across the phylogenetic tree were selected for experimental analysis in a heterologous host. Only members of group I could serve as receptors for class IIa bacteriocins, and the receptor efficiencies varied in a pattern directly related to their phylogenetic position. A multiple sequence alignment of IIC and IID proteins revealed three sequence regions (two in IIC and one in IID) that distinguish members of the bacteriocin-susceptible group from those of the other groups, suggesting that these amino acid regions confer the specific bacteriocin receptor function. Moreover, we demonstrated that variation in sensitivity might also exist within the same species due to differential expression levels of the receptor, since three strains ofLactobacillus sakeiharbouring identical man-PTSs were shown to display different expression levels of a man-PTS gene that corresponded to the variation in bacteriocin sensitivity. Together, the results of our study show that the level of bacteriocin susceptibility for a bacterial cell is primarily determined by differences in its man-PTS proteins, although the expression levels of the corresponding genes also play an important role.
Effects of oriC relocation on control of replication initiation in Bacillus subtilis
Authors: Moriya Shigeki , Kawai Yoshikazu , Kaji Sakiko , Smith Adrian , Harry Elizabeth J. , Errington Jeffery
Published: August 2009

In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. InEscherichia coli, positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle. In the Gram-positive bacteriumBacillus subtilis, much less is known about the regulation of replication initiation, specifically, regarding negative control mechanisms which ensure replication initiation only once per cell cycle. Here we report that replication initiation was greatly enhanced in strains that had the origin of replication (oriC) relocated to various loci on the chromosome. WhenoriCwas relocated to new loci further than 250 kb counterclockwise from the native locus, replication initiation became asynchronous and earlier than in the wild-type cells. In twooriC-relocated strains (oriCatargGorpnbA, 25 ° or 30 ° on the 36 ° chromosome map, respectively), DnaA levels were higher than in the wild-type but not enough to cause earlier initiation of replication. Our results suggest that the initiation capacity of replication is accumulated well before the actual time of initiation, and its release may be suppressed by a unique DNA structure formed near the nativeoriClocus.
Chlamydia trachomatis YtgA is an iron-binding periplasmic protein induced by iron restriction
Authors: Miller J. D. , Sal M. S. , Schell M. , Whittimore J. D. , Raulston J. E.
Published: August 2009

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is the causative agent of common sexually transmitted diseases and the leading cause of preventable blindness worldwide. It has been observed that YtgA (CT067) is very immunogenic in patients with chlamydial genital infections. Homology analyses suggested that YtgA is a soluble periplasmic protein and a component of an ATP-binding cassette (ABC) transport system for metals such as iron. Since little is known about iron transport in C. trachomatis, biochemical assays were used to determine the potential role of YtgA in iron acquisition. 59Fe binding and competition studies revealed that YtgA preferentially binds iron over nickel, zinc or manganese. Western blot and densitometry techniques showed that YtgA concentrations specifically increased 3–5-fold in C. trachomatis, when cultured under iron-starvation conditions rather than under general stress conditions, such as exposure to penicillin. Finally, immuno-transmission electron microscopy provided evidence that YtgA is more concentrated in C. trachomatis during iron restriction, supporting a possible role for YtgA as a component of an ABC transporter.
Identification of two [4Fe–4S]-cluster-containing hydro-lyases from Pyrococcus furiosus
Authors: van Vugt-Lussenburg Barbara M. A. , van der Weel Laura , Hagen Wilfred R. , Hagedoorn Peter-Leon
Published: August 2009

The hyperthermophilic archaeonPyrococcus furiosusis a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely relatedPyrococcus horikoshiiandPyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on thePyr. furiosusgenome. In the present study, these three genes were heterologously overexpressed inEscherichia colito enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe–4S]-cluster-containing heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe–S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe–4S]-cluster-containing fumarate hydratases exemplified by MmcBC fromPelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase inPyr. furiosushas yet to be established.
Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements
Authors: Sampson Timothy , Broussard Gregory W. , Marinelli Laura J. , Jacobs-Sera Deborah , Ray Mondira , Ko Ching-Chung , Russell Daniel , Hendrix Roger W. , Hatfull Graham F.
Published: August 2009

Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41 901 bp, 42 289 bp and 41 441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.
Proteolytic degradation of human salivary MUC5B by dental biofilms
Authors: Wickström Claes , Herzberg Mark C. , Beighton David , Svensäter Gunnel
Published: August 2009

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14–40×106 Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.
Identification and characterization of a family of toxin–antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module
Authors: Weaver Keith E. , Reddy Shirisha G. , Brinkman Cassandra L. , Patel Smita , Bayles Kenneth W. , Endres Jennifer L.
Published: August 2009

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.
Interactions of Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria with epithelial and phagocytic cells
Authors: Saldías M. Soledad , Valvano Miguel A.
Published: August 2009

Burkholderia cenocepacia is a member of the B. cepacia complex (Bcc), a group of opportunistic bacteria that infect the airways of patients with cystic fibrosis (CF) and are extraordinarily resistant to almost all clinically useful antibiotics. Infections in CF patients with Bcc bacteria generally lead to a more rapid decline in lung function, and in some cases to the ‘cepacia syndrome’, a virtually deadly exacerbation of the lung infection with systemic manifestations. These characteristics of Bcc bacteria contribute to higher morbidity and mortality in infected CF patients. In the last 10 years considerable progress has been made in understanding the interactions between Bcc bacteria and mammalian host cells. Bcc isolates can survive either intracellularly within eukaryotic cells or extracellularly in host tissues. They survive within phagocytes and respiratory epithelial cells, and they have the ability to breach the respiratory epithelium layer. Survival and persistence of Bcc bacteria within host cells and tissues are believed to play a key role in pulmonary infection and to contribute to the persistent inflammation observed in patients with CF. This review summarizes recent findings concerning the interaction between Bcc bacteria and epithelial and phagocytic cells.
Extraction of RNA from Amphioxus Embryos or Adult Amphioxus Tissue
Authors: Yu J.K. Sky , Holland Linda Z.
Published: September 2009

INTRODUCTION

This protocol describes how to isolate total RNA from amphioxus using a patented TRI reagent that is commercially available from several sources. The protocol can be applied to both adult tissues and embryos, although we recommend different sample pretreatment methods for adults and embryos. After the samples are prepared, they are homogenized in TRI reagent, which disrupts the cells and maintains the integrity of RNA. After the addition of chloroform and phase separation, RNA is extracted in the aqueous phase and subsequently recovered by precipitation with isopropyl alcohol. The resulting RNA can be used for gel blotting (Northern analysis), selection of polyadenylated RNA, or for synthesizing cDNA with reverse transcriptase PCR (RT-PCR).
Fluorescent In Situ Hybridization (FISH) on Diploid Nuclei and Mitotic Chromosomes from Drosophila melanogaster Larval Tissues
Authors: Blattes Roxane , Käs Emmanuel
Published: September 2009

INTRODUCTION

This protocol adapts the multicolor fluorescent in situ hybridization (FISH) method for use with interphase nuclei and mitotic chromosomes of neuroblasts from dissected Drosophila melanogaster larval brains. It is suitable for the simultaneous detection of two or more target sequences, is readily applicable to heterochromatin sequences, and provides the specificity and sensitivity needed to covisualize single-copy and repeated sequences. It is equally successful for detecting other sequences, such as single-copy euchromatin genes. This is made possible chiefly through the use of fixation procedures adjusted to suit the nature of the hybridization targets (e.g., condensed heterochromatin sequences), as well as the use of a mild chemical treatment to denature DNA instead of heat. Also, omitting mitotic blockers such as colchicine yields chromosomes with well-preserved morphology and permits detailed mapping of detected sequences. Most importantly, using this technique on diploid mitotic chromosomes and interphase nuclei allows the unambiguous localization of under-replicated heterochromatin sequences that cannot be resolved within the chromocenter of polytene chromosomes.
Tissue Sampling and Genomic DNA Purification from the Western Clawed Frog Xenopus tropicalis: Figure 1.
Authors: Showell Chris , Conlon Frank L.
Published: September 2009

INTRODUCTION

The extraction of genomic DNA from Western clawed frog (Xenopus tropicalis) tissue samples is an essential step for genotyping known mutations, identifying live animals carrying nonfluorescent transgenes, and reverse genetic screening techniques. We describe here a method for sampling tail tissue from X. tropicalis tadpoles at stage 48 and later. This method of tissue sampling allows a significant amount of genomic DNA to be obtained from tadpoles without killing them. In both Xenopus laevis and X. tropicalis, the tip of the tail is able to regenerate following surgery and its removal does not have a significant effect on the survival of the tadpole when performed carefully. Genomic DNA purification can be carried out in “deep-well” 96-well plates, making it amenable to high-throughput applications. Typically, the DNA yield is sufficient for more than 100 standard 50-μL polymerase chain reactions (PCRs) (using 1-5 μL of DNA per reaction), so it can be used for genetic screening and mapping studies.
Evaluation of the capacity of three halophytes to desalinize their rhizosphere as grown on saline soils under nonleaching conditions
Authors: Rabhi Mokded , Hafsi Chokri , Lakhdar Abdelbasset , Hajji Saïd , Barhoumi Zouhaier , Hamrouni Mohamed Hédi , Abdelly Chedly , Smaoui Abderrazak
Published: August 2009

In the sabkha of Soliman (N‐E Tunisia), soil samples of the upper 20 cm were taken during the driest period of the year (July–August) from inside and outside tufts of two perennial halophytes: Arthrocnemum indicum (Willd.) Moq. and Suaeda fruticosa Forssk., both from family Chenopodiaceae. Samples were analysed for electrical conductivity of the saturation paste extract (ECe) and soluble sodium (Na+) content. Then, tufts were divided into three size categories and their shoot biomass production and Na+ content were determined. Our results showed a considerable contribution of shoot Na+ accumulation to rhizosphere desalination. The capacity of the two native halophytes A. indicum. and S. fruticosa to desalinize saline soils was compared with that of an introduced halophyte, Sesuvium portulacastrum L. (Aizoaceae). Seedlings were grown under greenhouse conditions in pots containing 8 kg of saline soil each. Pots were irrigated with tap water during 170 days without leaching. Our results confirmed the contribution of shoot Na+ accumulation to soil desalination. They showed also that among the three studied species, Sesuvium portulacastrum L. seems to be the most convenient to be used for this purpose in arid and semi‐arid regions where precipitation is too low to leach salts from rhizosphere.
The structure and status of forest fragments outside protected areas in central Uganda
Authors: Baranga Deborah , Chapman Colin A. , Kasenene John M.
Published: August 2009

Given the extent of tropical forest deforestation and as a number of conservation programmes and local communities rely on forest fragments, it has become important to understand how fragment exploitation by local communities affects forest structure and function. The effects of forest exploitation on forest structure and status of forest fragments were investigated in 20 nonreserved forest fragments in central Uganda. Enumeration of plots showed that tree species composition of the forest fragments was 60.0%, 23.7% and 6.3% for under‐storey, middle and top canopy trees respectively. The major activity was fuel wood extraction (65%), followed by brick making (10%), cultivation and livestock paddocks (10%), charcoal burning (5%), local brew distillation and others (5%). These extractive processes caused drastic structural changes, habitat degradation and destruction. Tree stumps enumeration indicated that under storey trees formed the highest proportion for wood extraction. There was no significant difference in the level of forest exploitation (basal area loss) among forest patches of varying sizes.
The ecological impacts of a migratory bat aggregation on its seasonal roost in Kasanka National Park, Zambia
Authors: Byng James W. , Racey Paul A. , Swaine Mike D.
Published: August 2009

The ecological impacts of migratory species on their seasonal environments are poorly known. The effects of several million straw‐coloured fruit bats (Eidolon helvum), which migrate seasonally to Kasanka National Park, Zambia each year, on the small area of mushitu‘swamp’ forest in which they roost, were investigated. The structure of the mushitu forest was profoundly altered by the presence of E. helvum, with increased roost tree mortality, lowering and opening of the forest canopy and a decrease in tree basal area. Eidolon helvum are also thought to increase the severity of fires within their roost site because the structural changes result in a drier microclimate. The effects of increased nitrogen and phosphorous input were less apparent. These findings suggest that the ecological impact of this migratory bat species on its seasonal environment could ultimately threaten the long‐term viability of its seasonal roost.
Environmental parameters and anthropogenic effects predicting the spatial distribution of wild ungulates in the Akagera savannah ecosystem
Authors: Averbeck Christiane , Apio Ann , Plath Martin , Wronski Torsten
Published: August 2009

Savannah areas affected by human activities such as livestock keeping and agriculture are often characterized by shifts in landscape structuring, with a predominance of few(er) habitat types. This is typically accompanied by pronounced changes in the communities of ungulates. The aim of this study was to find out whether shifts in ungulate communities in Lake Mburo National Park (LMNP) are primarily predicted by an alteration in the composition of the preferred habitat types or if more complex interactions between habitat changes and the prevalence of ungulates occur. Monthly road counts were used to establish the number of eleven ungulate species in LMNP and adjacent unprotected Ankole Ranching Scheme. The common duiker (Sylvicapra grimmia campbelliae Gray, 1843) was found in more abundance in disturbed areas, while showing a significant change in habitat use. Common duiker tended to use the vegetation type otherwise used by the bushbuck (Tragelaphus scriptus dama Neumann, 1902). Our results support the claim that the occurrence of ungulates is not only directly affected by the availability of ‘suitable’ habitats, but behavioural plasticity and competitive exclusion also need to be considered.
Space use of the spotted sand lizard (Pedioplanis l. lineoocellata) under different degradation states
Authors: Wasiolka Bernd , Jeltsch Florian , Henschel Joh , Blaum Niels
Published: August 2009

Although the effects of grazing‐induced savannah degradation on animal diversity are well documented, knowledge of how they affect space use or responding behaviour remains poor. In this study, we analysed space use of the spotted sand lizard (Pedioplanis l. lineoocellata) in degraded versus nondegraded habitats of southern Kalahari savannah habitats. Lizards were radio tracked, daily movement distances recorded and home range sizes calculated. In degraded Kalahari savannah habitats where plant diversity and perennial grass cover are low but shrub cover high, P. lineoocellata moves larger distances (40.88 ± 6.42 m versus 27.43 ± 5.08 m) and occupies larger home ranges (646.64 ± 244.84 m2 versus 209.15 ± 109.84 m2) than in nondegraded habitats (high plant diversity, high perennial grass cover and low shrub cover). We assume that this increase in daily movement distances and home range sizes is a behavioural plasticity to limited food resources in degraded savannah habitats. Although P. lineoocellata is able to adjust to resource‐poor savannah habitats, the increase in the lizard’s movement activities is likely to result in a higher predation risk. This is supported by the lower availability of protective vegetation i.e. perennial grass cover. Hence, we conclude that despite behavioural plasticity of P. lineoocellata, overgrazing has a severe negative impact on the space use of P. lineoocellata.
Effect of polymorphisms in the FASN, OLR1, PPARGC1A, PRL and STAT5A genes on bovine milk‐fat composition
Authors: Schennink A. , Bovenhuis H. , Léon‐Kloosterziel K. M. , Van Arendonk J. A. M. , Visker M. H. P. W.
Published: August 2009

The aim of our study was to estimate effects of polymorphisms in the ATP‐binding cassette G2 (ABCG2), fatty acid synthase (FASN), oxidized low‐density lipoprotein receptor 1 (OLR1), peroxysome proliferator‐activated receptor‐γ coactivator‐1α (PPARGC1A), prolactin (PRL) and signal transducer and activator of transcription 5A (STAT5A) genes on milk production traits and detailed milk‐fat composition. Milk‐fat composition phenotypes were available for 1905 Dutch Holstein–Friesian cows. First, the presence of each SNP in the Dutch Holstein–Friesian population was evaluated by direct sequencing of the PCR product surrounding the SNP in 22 proven Dutch Holstein–Friesian bulls. The ABCG2 SNP did not segregate in the bull population. Second, we genotyped the cows for the FASNg.16024G>A, FASNg.17924A>G, OLR1g.8232C>A, PPARGC1Ac.1790+514G>A, PPARGC1Ac.1892+19G>A, PRLg.8398G>A and STAT5Ag.9501G>A polymorphisms, and estimated genotype effects on milk production traits and milk‐fat composition. FASNg.17924A>G and OLRg.8232C>A had a significant effect (P < 0.05) on milk‐fat percentage. However, we were not able to confirm results reported in the literature that showed effects of all evaluated polymorphisms on milk‐fat percentage or milk‐fat yield. All polymorphisms showed significant effects (P < 0.05) on milk‐fat composition. The polymorphisms in FASN and STAT5A, which had an effect on C14:0 and were located on chromosome 19, could not fully explain the quantitative trait locus for C14:0 that was previously detected on chromosome 19 in a genome‐wide scan using linkage analysis.