bluuebunny/crossref_metadata_2025 huggingface dataset:
Aggregate breakdown dynamics under rainfall compared with aggregate stability measurements
Authors: Legout C. , Leguédois S. , Le Bissonnais Y.
Published: March 2005

Aggregate breakdown due to rainfall action causes crusting and interrill erosion. Erodibility is seemingly determined by the capacity of surface aggregates to resist the effects of rainfall. In this paper, we evaluated the relevance of an aggregate stability measurement, which comprises three treatments, in order to characterize aggregate breakdown dynamics. Two cultivated soils were studied: a clay loam slightly sensitive to erosion and a more susceptible silt loam. We compared the size distributions of microaggregates produced by the three aggregate stability treatments with the results from a rainfall simulation. The behaviour of four initial aggregate size classes (< 3 mm, 3–5 mm, 5–10 mm and 10–20 mm) was also compared to study the influence of the initial aggregate size on the nature of resulting aggregates. The mean weight diameter was from 200 to 1400 µm for the silt loam and from 600 to 7000 µm for the clay loam. The two experiments – aggregate stability measurements and aggregate breakdown dynamics under rainfall – yielded similar results. Qualitative analysis showed that for both soils the sizes of fragments produced by breakdown with the aggregate stability tests and under rainfall were similar and seemed to be qualitatively independent of the size of initial aggregates. We first schematized the structural organization of aggregates in cultivated horizons with a simple hierarchical model at two levels: (i) < 250 µm microaggregates and (ii) > 250 µm macroaggregates made by the binding together of microaggregates. We then developed a model of aggregate breakdown dynamics under rainfall which gives, for various rainfall durations, the size distributions of resulting fragments on the basis of aggregate stability measurements. We obtained a correlation coefficient, r, of 0.87 for the silt loam and of 0.91 for the clay loam, showing that the experimental and predicted mass percentages were linearly related for each size fraction.
Contact angle of soils as affected by depth, texture, and land management
Authors: Woche S. K. , Goebel M.‐O. , Kirkham M. B. , Horton R. , Van der Ploeg R. R. , Bachmann J.
Published: March 2005

Water repellency can be a significant factor in soil physical behaviour, but little is known about the depth dependence of the contact angle of field soils. We investigated contact angles and wetting properties as a function of depth for a wide range of agricultural and forest soils in Germany. The agricultural soils ranged from silty to sandy texture (six profiles), and the forest soils ranged from sandy to loamy texture (eight profiles). Contact angles (CA) were measured with the Wilhelmy plate method (WPM). In most of the soils, advancing WPM contact angles were considerably greater than 0°  and they varied irregularly with depth. In general, sandy soils had larger WPM contact angles than silty soils. From the relation of the contact angle with texture and pH the quality of soil organic matter (SOM) was considered as more important for the wetting properties than the total amount of soil organic carbon (SOC). Finally, it was found that for soils with intermediate sand contents either under agricultural or forest use, the kind of land use seemed not to influence CA. Coarse‐textured sandy soils that were used only as forest sites were more hydrophobic than silty soils which were exclusively used as agricultural soils. We conclude that a coarse texture favours, in combination with other factors (mainly pH), hydrophobic SOM.
Biological and physical resilience of soil amended with heavy metal‐contaminated sewage sludge
Authors: Griffiths B. S. , Hallett P. D. , Kuan H. L. , Pitkin Y. , Aitken M. N.
Published: March 2005

Stability and resilience of a variety of soil properties and processes are emerging as key components of soil quality. We applied recently developed measures of biological and physical resilience to soils from an experimental site treated with metal‐contaminated sewage sludge. Soils treated with cadmium‐, copper‐ or zinc‐contaminated, digested or undigested sewage sludge were studied. Biological stability and resilience indices were: (i) the time‐dependent effects of either a transient stress (heating to 40°C for 18 hours) or a persistent stress (amendment with CuSO4) on decomposition, and (ii) the mineralization of dissolved organic carbon (DOC) released by drying–rewetting cycles. Physical stability and resilience measures were: (i) compression and expansion indices of the soils, and (ii) resistance to prolonged wetting and structural regeneration through drying–rewetting cycles. Soil total carbon and DOC levels were greater in the sludge‐amended soils, but there were no differential effects due to metal contamination of the sewage sludge. Effects of metals on physical resilience were greater than effects on soil C, there being marked reductions in the expansion indices with Cd‐ and Cu‐contaminated sludge, and pointed to changes in soil aggregation. The rate of mineralization of DOC released by drying and wetting was reduced by Zn contamination, while biological resilience was increased in the Zn‐contaminated soil and reduced by Cd contamination. We argue that physical and biological resilience are potentially coupled through the microbial community. This needs to be tested in a wider range of soils, but demonstrates the benefits from a combined approach to the biological and physical resilience of soils.
A multiscale study of silty soil structure
Authors: Bartoli F. , Genevois‐Gomendy V. , Royer J. J. , Niquet S. , Vivier H. , Grayson R.
Published: March 2005

Dependency of soil properties on scale is a crucial issue in soil physics. In this paper, fractal approaches are used in two case studies in France and Australia, respectively, to study how measured physical soil properties change with the sample spacing and the scale of observation. At a scale of 10–1000 m (104 to 106 mm), fractals were applied to sample data from a linear transect, while at the 10−6 to 102 mm scale, fractals were applied in two dimensions to analyse both soil micro‐ and macrostructure, based on thin section samples. Porosity was characterized by short‐range spatial variations using sample spacings of 0.5 and 5 m (from the transect data), and a sample spacing of 1 cm (from the thin section analysis). The size of the representative elementary volume (REV) or representative elementary area (REA), required to represent statistically the elementary soil structure, was identified in three ways: (i) by the correlation length of a representative interconnected pore network, (ii) by the upper limit of the non‐linear increase with observation scale of mean porosity (upper limit of the solid mass fractal domain), and (iii) by the non‐linear decrease with observation scale of the coefficient of variation, CV, of mean porosity. Two embedded REAs were identified: the first (0.1–0.4 mm) related to the soil microstructure whereas a second (11–44 mm) related to the soil macrostructure. The solid mass fractal dimensions of the two embedded structural domains showed that hierarchical heterogeneity of soil structure was more pronounced for microstructures than for macrostructures. The mean area ratio of microstructural matrix/total surface and the CV of mean microporosity both scale similarly at observation scales smaller than the REA size. Their scaling exponents were both related to the fractal dimension of microstructural matrix. This preliminary study shows that the theory of fractals applied to soil structures at a specific scale range cannot be directly applied to predict soil physical properties at another scale range. This is because there are different interdependent structuring processes operating at different scales resulting in fractal dimensions being consistent only over particular domain limits.
Forest vegetation does recycle substantial amounts of silicon from and back to the soil solution with phytoliths as an intermediate phase, contrary to recent reports
Authors: Farmer V. C.
Published: March 2005

Recent reports that forest vegetation takes up little Si and returns little to the soil in litter are based on the mistaken assumption that digestion of leaf and litter in hot oxidizing acids can bring phytoliths, a form of opaline silica, into solution. Phytolith formation and dissolution can explain a number of otherwise puzzling aspects of Si in soil solution.
Glucose‐dependent cell size is regulated by a G protein‐coupled receptor system in yeast Saccharomyces cerevisiae
Authors: Tamaki Hisanori , Yun Cheol‐Won , Mizutani Tomohiro , Tsuzuki Takahiro , Takagi Yukinobu , Shinozaki Makiko , Kodama Yukiko , Shirahige Katsuhiko , Kumagai Hidehiko
Published: March 2005

In the yeast, Saccharomyces cerevisiae, cell size is affected by the kind of carbon source in the medium. Here, we present evidence that the Gpr1 receptor and Gpa2 Gα subunit are required for both maintenance and modulation of cell size in response to glucose. In the presence of glucose, mutants lacking GPR1 or GPA2 gene showed smaller cells than the wild‐type strain. Physiological studies revealed that protein synthesis rate was reduced in the mutant strains indicating that reduced growth rate, while the level of mRNAs for CLN1, 2 and 3 was not affected in all strains. Gene chip analysis also revealed a down‐regulation in the expression of genes related to biosynthesis of not only protein but also other cellular component in the mutant strains. We also show that GPR1 and GPA2 are required for a rapid increase in cell size in response to glucose. Wild‐type cells grown in ethanol quickly increased in size by addition of glucose, while little change was observed in the mutant strains, in which glucose‐dependent cell cycle arrest caused by CLN1 repression was somewhat alleviated. Our study indicates that the yeast G‐protein coupled receptor system consisting of Gpr1 and Gpa2 regulates cell size by affecting both growth rate and cell division.
Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans
Authors: Ruksana Razia , Kuroda Kazuki , Terami Hiromi , Bando Tetsuya , Kitaoka Shun , Takaya Tomohide , Sakube Yasuji , Kagawa Hiroaki
Published: March 2005

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni‐1, tni‐2/unc‐27 and tni‐3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni‐4 was expressed solely in the pharynx. Expressing tni‐1 and ‐2 gene RNAi caused motility defects similar to unc‐27 (e155) mutant, a tni‐2 null allele. The tni‐3 RNAi expression produced egg laying defects while the tni‐4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI‐4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.
Phosphorylation of PKC activation loop plays an important role in receptor‐mediated translocation of PKC
Authors: Seki Takahiro , Matsubayashi Hiroaki , Amano Taku , Shirai Yasuhito , Saito Naoaki , Sakai Norio
Published: March 2005

Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation‐dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant γ‐ or δ‐PKC fused with GFP (γ‐ or δ‐PKC‐ALM (activation loop mutant)‐GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor‐mediated translocation with wild‐type PKC‐GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild‐type γ‐ or δ‐PKC‐GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. γ‐ or δ‐PKC‐ALM‐GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase‐negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+]i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC‐ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC‐ALM at the plasma membrane. It is possible that PKC‐ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor‐mediated PKC targeting.
Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication forks in Escherichia coli
Authors: Shibata Tatsuya , Hishida Takashi , Kubota Yoshino , Han Yong‐Woon , Iwasaki Hiroshi , Shinagawa Hideo
Published: March 2005

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA‐dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV‐induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature‐dependent growth defect of dnaE486 (Pol III α‐catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of ΔmgsA ΔrecA cells or the lethality of polA12 ΔrecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.
Effects of genetic ablation of bach1 upon smooth muscle cell proliferation and atherosclerosis after cuff injury
Authors: Omura Shinji , Suzuki Hiroshi , Toyofuku Mamoru , Ozono Ryoji , Kohno Nobuoki , Igarashi Kazuhiko
Published: March 2005

Bach1 is a transcriptional repressor of the cytoprotective enzyme heme oxygenase‐1 (HO‐1). Although HO‐1 protects against atherosclerosis, the function of Bach1 in this process is poorly understood. We isolated peritoneal macrophages and aortic smooth muscle cells (SMC) from wild‐type and bach1‐deficient mice. bach1‐deficient macrophages expressed increased levels of HO‐1 and showed elevated phagocytic activity when incubated with 0.75 µm microspheres. In SMC, bach1‐ablation resulted in increased expression of HO‐1 and decreased proliferation in bromodeoxyuridine incorporation assay as compared with wild‐type cells. The up‐regulated phagocytic activity and reduced SMC proliferation of bach1‐deficient cells were not restored by Zinc (II) protoporphyrin IX, an inhibitor of HO, suggesting that HO‐independent mechanisms are also involved in the regulation of phagocytosis of macrophages and proliferation of SMC by Bach1. In wild‐type mice, cuff placement around femoral artery caused pronounced intimal proliferation without affecting the media, thus resulting in intimal to medial (I/M) volume ratio of 65.6%. bach1‐deficient mice had less degree of intimal growth (I/M ratio of 45.6%). These results indicate that Bach1 plays a critical role in the regulation of HO‐1 expression, macrophage function, SMC proliferation and neointimal formation. Bach1 may regulate gene expression in these cells during inflammation and atherogenesis.
Studies of Schizosaccharomyces pombe TFIIE indicate conformational and functional changes in RNA polymerase II at transcription initiation
Authors: Hayashi Kazuhiro , Watanabe Tomomichi , Tanaka Aki , Furumoto Tadashi , Sato‐Tsuchiya Chiaki , Kimura Makoto , Yokoi Masayuki , Ishihama Akira , Hanaoka Fumio , Ohkuma Yoshiaki
Published: March 2005

The general transcription factor TFIIE plays essential roles in transcription by RNA polymerase II (PolII). Despite recent progress, the elucidation of its precise mechanisms including biological functions awaits further characterization. We report the isolation and characterization of Schizosaccharomyces pombe TFIIE (spTFIIE). Like human and other eukaryotic TFIIE proteins, spTFIIE consists of α and β subunits and the genes encoding both subunits are essential for viability. Chromatin immunoprecipitation assays demonstrated that spTFIIE localizes to promoters in vivo. Mutational analysis of the C‐terminal basic helix‐loop region of TFIIEβ, which is involved in the transition from transcription initiation to elongation, revealed that transcription‐defective mutants affected in this region are also cold sensitive. The spTFIIEβ subunit binds both spTFIIEβ and spTFIIEα but spTFIIEα binds only spTFIIEβ. These results indicate that TFIIE forms an α2β2 heterotetramer in which two αβ heterodimers are connected via β subunits. Further analysis of binding specificities showed that spTFIIEβ binds the Rpb2 and Rpb12 subunits of PolII, whereas spTFIIEα predominantly binds Rpb5, which is located at the clamp region and changes conformation upon transcription initiation.
The FWD1/β‐TrCP‐mediated degradation pathway establishes a ‘turning off switch’ of a Cdc42 guanine nucleotide exchange factor, FGD1
Authors: Hayakawa Makio , Kitagawa Hideo , Miyazawa Keiji , Kitagawa Masatoshi , Kikugawa Kiyomi
Published: March 2005

FWD1/β‐TrCP is the F‐box protein that functions as the receptor subunit of the SCFFWD1/β‐TrCP ubiquitin ligase and has been shown to be responsible for the degradation of important signaling molecules such as IκBs and β‐catenin. Protein substrates of FWD1/β‐TrCP contain a consensus DSGΨXS motif (where Ψ represents a hydrophobic residue and X represents any amino acid). Recognition by FWD1/β‐TrCP requires phosphorylation of the conserved serines in that motif. Here we show that FGD1, a Cdc42 guanine nucleotide exchange factor (GEF), is a novel target of the SCFFWD1/β‐TrCP ubiquitin ligase. A mutant FGD1 protein, FGD1(SA), in which both of the critical serine residues in the DSGΨXS motif have been replaced by alanines, does not interact with FWD1/β‐TrCP and exhibits increased stability. Morphological changes induced by wild‐type FGD1 (FGD1(WT)) are reduced by the co‐expression of SCFFWD1/β‐TrCP whereas those induced by FGD1(SA) are not affected. FGD1(SA)‐expressing cells show a higher level of cell motility than FGD1(WT)‐expressing cells. We present a novel ‘turning off’ mechanism for the inactivation of FGD1, an upstream regulator for Cdc42.
53BP2 induces apoptosis through the mitochondrial death pathway
Authors: Kobayashi Shinya , Kajino Shinichi , Takahashi Naoko , Kanazawa Satoshi , Imai Kenichi , Hibi Yurina , Ohara Hirotaka , Itoh Makoto , Okamoto Takashi
Published: March 2005

The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl‐2, and p65 subunit of nuclear factor κB (NF‐κB). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans‐membrane potential (ΔΨm) and activation of caspase‐9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.
A new release on life: emerging concepts in proteolysis and parasite invasion
Authors: Carruthers Vern B. , Blackman Michael J.
Published: March 2005

Cell invasion by apicomplexan pathogens such as the malaria parasite and Toxoplasma is accompanied by extensive proteolysis of zoite surface proteins (ZSPs) required for attachment and penetration. Although there is still little known about the proteases involved, a conceptual framework is emerging for the roles of proteolysis in cell invasion. Primary processing of ZSPs, which includes the trimming of terminal peptides or segmentation into multiple fragments, is proposed to activate these adhesive ligands for tight binding to host receptors. Secondary processing, which occurs during penetration, results in the shedding of ZSPs by one of two mechanistically distinct ways, shaving or capping. Resident surface proteins are typically shaved from the surface whereas adhesive ligands mobilized from intracellular secretory vesicles are capped to the posterior end of the parasite before being shed during the final steps of penetration. Intriguingly, recent studies have revealed that ZSPs can be released either by being cleaved adjacent to the membrane anchor or actually within the membrane itself. Mounting evidence suggests that intramembrane cleavage is catalysed by one or more integral membrane serine proteases of the Rhomboid family and we propose that several malaria adhesive ligands may be potential substrates for these enzymes. We also discuss the evidence that the key reason for ZSP shedding during invasion is to break the connection between parasite surface ligands and host receptors. The sequential proteolytic events associated with invasion by pathogenic protozoa may represent vulnerable pathways for the future development of synergistic anti‐protozoal therapies.
Mutant forms of the Escherichia coliβ sliding clamp that distinguish between its roles in replication and DNA polymerase V‐dependent translesion DNA synthesis
Authors: Sutton Mark D. , Duzen Jill M. , Maul Robert W.
Published: March 2005

The Escherichia coliβ sliding clamp is proposed to play an important role in regulating DNA polymerase traffic at the replication fork. As part of an ongoing effort to understand how organisms manage the actions of their multiple DNA polymerases, we examined the ability of several mutant forms of the β clamp to function in DNA polymerase V‐ (pol V‐) dependent translesion DNA synthesis (TLS) in vivo. Our results indicate that a dnaN159 strain, which expresses a temperature sensitive form of the β clamp, was impaired for pol V‐dependent TLS at the permissive temperature of 37°C. This defect was complemented by a plasmid that expressed near‐physiological levels of the wild‐type clamp. Using a dnaN159 mutant strain, together with various plasmids expressing mutant forms of the clamp, we determined that residues H148 through R152, which comprise a portion of a solvent exposed loop, as well as position P363, which is located in the C‐terminal tail of the β clamp, are critically important for pol V‐dependent TLS in vivo. In contrast, these same residues appear to be less critical for pol III‐dependent replication. Taken together, these findings indicate that: (i) the β clamp plays an essential role in pol V‐dependent TLS in vivo and (ii) pol III and pol V interact with non‐identical surfaces of the β clamp.
Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum
Authors: Doan Thierry , Marquis Kathleen A. , Rudner David Z.
Published: March 2005

During the process of spore formation in Bacillus subtilis many membrane proteins localize to the sporulation septum where they play key roles in morphogenesis and cell‐cell signalling. However, the mechanism by which these proteins are anchored at this site is not understood. In this report we have defined the localization requirements for the mother‐cell membrane protein SpoIVFA, which anchors a signalling complex in the septal membrane on the mother cell side. We have identified five proteins (SpoIID, SpoIIP, SpoIIM, BofA and SpoIIIAH) synthesized in the mother cell under the control of σE and one protein (SpoIIQ) synthesized in the forespore under the control of σF that are all required for the proper localization of SpoIVFA. Surprisingly, these proteins appear to have complementary and overlapping anchoring roles suggesting that SpoIVFA is localized in the septal membrane through a web of protein interactions. Furthermore, we demonstrate a direct biochemical interaction between the extracellular domains of two of the proteins required to anchor SpoIVFA: the forespore protein SpoIIQ and the mother‐cell protein SpoIIIAH. This result supports the idea that the web of interactions that anchors SpoIVFA is itself held in the septal membrane through a zipper‐like interaction across the sporulation septum. Importantly, our results suggest that a second mechanism independent of forespore proteins participates in anchoring SpoIVFA. Finally, we show that the dynamic localization of SpoIIQ in the forespore is impaired in the absence of SpoIVFA but not SpoIIIAH. Thus, a complex web of interactions among mother cell and forespore proteins is responsible for static and dynamic protein localization in both compartments of the sporangium. We envision that this proposed network is involved in anchoring other sporulation proteins in the septum and that protein networks with overlapping anchoring capacity is a feature of protein localization in all bacteria.
Maturation of the Escherichia coli divisome occurs in two steps
Authors: Aarsman Mirjam E. G. , Piette André , Fraipont Claudine , Vinkenvleugel Thessa M. F. , Nguyen‐Distèche Martine , den Blaauwen Tanneke
Published: March 2005

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno‐ and GFP‐fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14–21 min, depending on the growth rate, between Z‐ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two‐step model for bacterial division in which the Z‐ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.
Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence
Authors: Narasipura Srinivas D. , Chaturvedi Vishnu , Chaturvedi Sudha
Published: March 2005

We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36–66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth‐defective at 37°C in 19–20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37°C as well at 30°C in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O2• −) in the mitochondria. The sod2 was also highly susceptible to redox‐cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.
Evidence for non‐enzymatic glycosylation of Escherichia coli chromosomal DNA
Authors: Mironova Roumyana , Niwa Toshimitsu , Handzhiyski Yordan , Sredovska Angelina , Ivanov Ivan
Published: March 2005

We have recently shown that the process of non‐enzymatic glycosylation (glycation) takes place in Escherichia coli under physiological conditions and affects both recombinant and endogenous bacterial proteins. In this study, we further demonstrate that E. coli chromosomal DNA is also subjected to glycation under physiological growth conditions. The E. coli DNA accumulates early glycation (Amadori) products as proven by the nitroblue tetrazolium (NBT) reduction assay. It showed also immunoreactivity to a monoclonal antibody raised against N∈‐(carboxymethyl)lysine and fluorescent properties indicative of modifications with advanced glycation end‐products. Two types of fluorophores were detected in the E. coli DNA with excitation maxima at 360 nm and 380 nm and emission maxima at 440 nm and 410 nm. Using the NBT reduction assay, fluorescence spectroscopy and enzyme‐linked immunosorbent assay we revealed that glycation adducts accumulate in DNA predominantly in the stationary phase of growth, although they could be detected also in exponential‐phase cells. Besides on the growth phase, the extent of DNA glycation depends also on the nutrient broth composition being more extensive in rich media. Thiamine was found to inhibit both DNA glycation and spontaneous point mutations as judged by the decreased rate of the argE3 to Arg+ reversions in the E. coli strain AB1157.
The expanding world of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus
Authors: Zago Maria A. , Dennis Patrick P. , Omer Arina D.
Published: March 2005

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K‐turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae‐associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two‐thirds of these sequences possessed K‐turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5′ or 3′ ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.
A magnesium‐dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis
Authors: Formstone Alex , Errington Jeffery
Published: March 2005

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod‐shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co‐transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in‐frame deletion of mreB and its complementation by mreB+ only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP–MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.
Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ
Authors: Crepin Valérie F. , Prasannan Sunil , Shaw Rob K. , Wilson Rebecca K. , Creasey Elizabeth , Abe Cecilia M. , Knutton Stuart , Frankel Gad , Matthews Steve
Published: March 2005

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram‐negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec‐dependent manner, and to bridge the TTSS membrane‐associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 – amino acid residues 1–55 in the mature protein, and D2 – amino acid residues 90–170), each comprising a three‐stranded β‐sheet flanked by two α‐helices. A flexible region (residues 60–85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJD1 and EscJD2 in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJD1 and EscJD2 in the same wild‐type bacterium had a dominant‐negative phenotype suggesting defective assembly of the TTSS and protein translocation.
The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol
Authors: Colangeli Roberto , Helb Danica , Sridharan Sudharsan , Sun Jingchuan , Varma‐Basil Mandira , Hazbón Manzour Hernando , Harbacheuski Ryhor , Megjugorac Nicholas J. , Jacobs William R. , Holzenburg Andreas , Sacchettini James C. , Alland David
Published: March 2005

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first‐line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR‐pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two‐dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.
Unique mechanistic features of post‐translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability
Authors: Ehlers Claudia , Weidenbach Katrin , Veit Katharina , Forchhammer Karl , Schmitz Ruth A.
Published: March 2005

PII‐like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII‐like protein, representing a novel PII receptor. The GlnK1 protein of the methanogenic archaeon Methanosarcina mazei strain Gö1 interacts and forms stable complexes with glutamine synthetase (GlnA1). Complex formation with GlnK1 in the absence of metabolites inhibits the activity of GlnA1. On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2‐oxoglutarate. Moreover, 2‐oxoglutarate antagonized the inhibitory effects of GlnK1 on GlnA1 activity but did not prevent GlnK1/GlnA1 complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2‐oxoglutarate, the nitrogen sensor GlnK1 allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2‐oxoglutarate stimulate maximal GlnA1 activity and transform GlnA1 into an activated conformation, which prevents inhibition by GlnK1. (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA1 activity is reduced by decreasing internal 2‐oxoglutarate concentrations through diminished direct activation and by GlnK1 inhibition.
A pathway‐specific transcriptional regulatory gene for nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development
Authors: Liu Gang , Tian Yuqing , Yang Haihua , Tan Huarong
Published: March 2005

DNA sequence analysis of a 7.5 kb XhoI DNA fragment from the region flanking the nikkomycin biosynthesis gene cluster in Streptomyces ansochromogenes revealed one 3.3 kb open reading frame (ORF), designated sanG. The deduced product of sanG (1061 amino acids), which is similar to PimR of Streptomyces natalensis, contains an OmpR‐like DNA binding domain in its N‐terminal portion and A‐ and B‐type nucleotide binding motifs in the middle of the protein. Disruption of sanG abolished nikkomycin biosynthesis, reduced sporulation and led to brown pigment accumulation. All aspects of this complex phenotype were complemented by a single copy sanG which was integrated into the chromosome. The introduction of multiple copies of sanG resulted in increased nikkomycin production. S1 mapping results indicated that sanG is transcribed from at least three promoters (P1, P2 and P3), P1 being strongly upregulated when production of nikkomycins starts. Two putative transcription units for nikkomycin biosynthesis, starting from sanN and sanO, are dependent on the expression of sanG, whereas a putative transcription unit starting from sanF was not regulated by sanG. These results suggested that sanG encodes a transcriptional activator important for nikkomycin biosynthesis that, unusually, also has pleiotropic effects on secondary metabolism and development.
A new in vivo termination function for DNA polymerase I of Escherichia coli K12
Authors: Markovitz Alvin
Published: March 2005

Escherichia coli deleted for the tus gene are viable. Thus there must be at least one other mechanism for terminating chromosome synthesis. The tus deletion strain yielded a small fraction of cells that overproduce DNA, as determined by flow cytometry after run‐out chromosome replication in the presence of rifampicin and cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only when arabinose was added to the medium to induce the synthesis of the Tus protein. Transduction studies were done to test whether or not additional chromosomal deletions could enhance the excess chromosome replication in the tus deletion strain. A strain containing a second deletion in metE udp overproduced DNA at a high level during run‐out replication. Further studies demonstrated that a spontaneous unknown mutation had occurred during the transduction. This mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D) alone into the tus deletion strain produced the high DNA overproduction phenotype. The polA(G544D) and six other polA alleles were then tested in wild‐type and in tus deletion backgrounds. The two alleles with low levels of 5′→3′ exonuclease (exo) overproduced DNA while those with either high or normal exo overproduce much less DNA in run‐out assays in the wild‐type background. In contrast, all seven mutant polA alleles caused the high DNA overproduction phenotype in a tus deletion background. To explain these results we propose a new in vivo function for wild‐type DNA polymerase I in chromosome termination at site(s) not yet identified.
Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin‐dependent C‐1 oxidation/reduction pathway and differences in H2 metabolism between closely related species
Authors: Guss Adam M. , Mukhopadhyay Biswarup , Zhang Jun Kai , Metcalf William W.
Published: March 2005

A mutation in the mch gene, encoding the enzyme 5,10‐methenyl tetrahydromethanopterin (H4MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H2/CO2, methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H2/CO2, demonstrating its ability to utilize H2 as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H2/CO2 is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H2/CO2, could not be made in wild‐type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H2. M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H2 oxidation. Interestingly, the ech‐dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H2‐dependent methyl reduction.
Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation‐associated protein by staphylococcal and host proteases
Authors: Rohde Holger , Burdelski Christoph , Bartscht Katrin , Hussain Muzaffar , Buck Friedrich , Horstkotte Matthias A. , Knobloch Johannes K.‐M. , Heilmann Christine , Herrmann Mathias , Mack Dietrich
Published: March 2005

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device‐related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA‐negative strains are not uncommon, suggesting the existence of PIA‐independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation‐associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap‐negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide‐independent manner. In contrast, expression of full‐length Aap did not lead to a biofilm‐positive phenotype. Obviously, to gain adhesive function, full‐length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by α2‐macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein‐dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.