bluuebunny/crossref_metadata_2025 huggingface dataset:
Poor response of macadamia (Macadamia integrifolia Maiden and Betche) to irrigation in a high rainfall area of subtropical Australia
Authors: Trochoulias T , Johns GG
Published: December 2005

Bearing macadamia trees (cv. Keauhou 246), received varying rates of irrigation at weekly intervals over 8 years, and the effects on nut yield and tree size were measured. Annual rainfall ranged between 1232 and 2283 mm and was supplemented by 9-24 irrigations per year. Unsatisfied evaporative demand in the control treatment was estimated to vary from 1 to 380 mm per season. Canopy and trunk areas were not affected by irrigation. The average annual yield per unit of canopy area across all treatments was about 900 g/m2 of nut in shell. Irrigation reduced nut in shell yield per unit trunk area slightly, and depressed the individual mean weight of nut in shell by an average of 5%. There was a highly significant (P<0.01) inverse linear relationship between individual nut weight and irrigation amount, with an individual mean nut in shell reduction of 7% at the highest irrigation rate. Kernel weight, as a percentage of total nut in shell weight (kernel recovery), was not affected by irrigation, but the percentage of kernels that floated in tap water (grade 1 kernels) was 2.8% higher from control trees than from irrigated trees.
Use of high electrical conductivity of nutrient solution to improve the quality of salad tomatoes (Lycopersicon esculentum) grown in hydroponic culture
Authors: Cornish PS
Published: December 2005

The effect of the electrical conductivity (EC) of nutrient solution on total soluble solids (TSS) and fruit yield was studied, using table tomatoes grown outdoors in hydroponic culture, with the aim of seeing if salt stress could be used to increase TSS and thereby improve fruit quality. Two initial experiments compared the responses of 3 different cultivars and compared responses to different salts. The main experiment aimed to quantify the trade-off between yield and TSS as EC was raised with NaCl. In the main experiment, TSS of cv. Flora Dade increased linearly from 4.40 to 5.94 �Brix over an EC range of 1.5-9.0 mS/cm. Over all 3 experiments, the mean TSS of Flora Dade increased by 0.16 �Brix/mS.cm rise in EC above the control EC of 1.5-2.0 mS/cm. The 3 cultivars responded similarly to raised EC, although they ranged in TSS from 4.1 to 5.4 �Brix under nonsaline conditions. Raising solution EC with NaCl, KCl, or a mixture of NaCl/CaCl2 all gave significant increases in TSS. Increasing EC also increased titratable acidity and reduced fruit size, but had no effect on fruit firmness. Yield in the main experiment declined with increasing EC, but not linearly. Over all experiments, it was shown that salt stress could be used to achieve an increase in TSS of about 0.5 �Brix with little or no effect on yield. However, the EC at which this increase occurred appeared to depend on weather conditions.
Incidence of Alternaria brassicicola (Schw.) Wiltsh. on Brassica oleracea seeds
Authors: Sivapalan A , Browning JW
Published: December 2005

Samples of Brassica oleracea seed from Victoria, were tested for the presence of seed-borne Alternaria brassicicola and Alternaria brassicae. A. brassicicola was detected in 26 of 44 samples tested but A. brassicae was not detected in any. Between 24 and 37% of seed was infected, with 4-8% of infection found in the embryo tissues. Inoculation of seed with A. brassicicola resulted in loss of vigour in germinated seedlings, followed by death. The fungus retained its viability and pathogenicity on seed stored for up to 12 months. This investigation indicates that a high proportion of commercially available brassica seed are contaminated with A. brassicicola and may therefore be a primary source of disease for brassica crops in Australia.
Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis
Authors: van Staveren Wilma C. G. , Solís David Weiss , Delys Laurent , Venet David , Cappello Matteo , Andry Guy , Dumont Jacques E. , Libert Frédérick , Detours Vincent , Maenhaut Carine
Published: December 2005

The cAMP signaling pathway regulates growth of many cell types, including somatotrophs, thyrocytes, melanocytes, ovarian follicular granulosa cells, adrenocortical cells, and keratinocytes. Mutations of partners from the cAMP signaling cascade are involved in tumor formation. Thyroid-stimulating hormone (TSH) receptor and Gsα activating mutations have been detected in thyroid autonomous adenomas, Gsα mutations in growth hormone-secreting pituitary adenomas, and PKAR1A mutations in Carney complex, a multiple neoplasia syndrome. To gain more insight into the role of cAMP signaling in tumor formation, human primary cultures of thyrocytes were treated for different times (1.5, 3, 16, 24, and 48 h) with TSH to characterize modulations in gene expression using cDNA microarrays. This kinetic study showed a clear difference in expression, early (1.5 and 3 h) and late (16–48 h) after the onset of TSH stimulation. This result suggests a progressive sequential process leading to a change of cell program. The gene expression profile of the long-term stimulated cultures resembled the autonomous adenomas, but not papillary carcinomas. The molecular phenotype of the adenomas thus confirms the role of long-term stimulation of the TSH–cAMP cascade in the pathology. TSH induced a striking up-regulation of different negative feedback modulators of the cAMP cascade, presumably insuring the one-shot effect of the stimulus. Some were down- or nonregulated in adenomas, suggesting a loss of negative feedback control in the tumors. These results suggest that in tumorigenesis, activation of proliferation pathways may be complemented by suppression of multiple corresponding negative feedbacks, i.e., specific tumor suppressors.
The up-regulation of ferritin expression using a small-molecule ligand to the native mRNA
Authors: Tibodeau Jennifer D. , Fox Paige M. , Ropp Patricia A. , Theil Elizabeth C. , Thorp H. Holden
Published: December 2005

The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE–yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.
Recombination, rearrangement, reshuffling, and divergence in a centromeric region of rice
Authors: Ma Jianxin , Bennetzen Jeffrey L.
Published: December 2005

Centromeres have many unusual biological properties, including kinetochore attachment and severe repression of local meiotic recombination. These properties are partly an outcome, partly a cause, of unusual DNA structure in the centromeric region. Although several plant and animal genomes have been sequenced, most centromere sequences have not been completed or analyzed in depth. To shed light on the unique organization, variability, and evolution of centromeric DNA, detailed analysis of a 1.97-Mb sequence that includes centromere 8 (CEN8) of
japonica
rice was undertaken. Thirty-three long-terminal repeat (LTR)-retrotransposon families (including 11 previously unknown) were identified in the CEN8 region, totaling 245 elements and fragments that account for 67% of the region. The ratio of solo LTRs to intact elements in the CEN8 region is ≈0.9:1, compared with ≈2.2:1 in noncentromeric regions of rice. However, the ratio of solo LTRs to intact elements in the core of the CEN8 region (≈2.5:1) is higher than in any other region investigated in rice, suggesting a hotspot for unequal recombination. Comparison of the CEN8 region of
japonica
and its orthologous segments from
indica
rice indicated that ≈15% of the intact retrotransposons and solo LTRs were inserted into CEN8 after the divergence of
japonica
and
indica
from a common ancestor, compared with ≈50% for previously studied euchromatic regions. Frequent DNA rearrangements were observed in the CEN8 region, including a 212-kb subregion that was found to be composed of three rearranged tandem repeats. Phylogenetic analysis also revealed recent segmental duplication and extensive rearrangement and reshuffling of the CentO satellite repeats.
Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source
Authors: Ronen Michal , Botstein David
Published: December 2005

To understand the dynamics of transcriptional response to changing environments, well defined, easily controlled, and short-term perturbation experiments were undertaken. We subjected steady-state cultures of
Saccharomyces cerevisiae
in chemostats growing on limiting galactose to two different size pulses of glucose, well known to be a preferred carbon source. Although these pulses were not large enough to change growth rates or cell size, ≈25% of the genes changed their expression at least 2-fold. Using DNA microarrays to estimate mRNA abundance, we found a number of distinguishable patterns of transcriptional response among the many genes whose expression changed. Many of these genes were already known to be regulated by particular transcription factors; we estimated five potentially relevant transcription factor activities from the observed changes in gene expression (i.e., Mig1p, Gal4p, Cat8p, Rgt1p, Adr1p, and Rcs1p). With these estimates, for two regulatory circuits involving interaction among multiple regulators we could generate dynamical models that quantitatively account for the observed transcriptional responses to the transient perturbations.
The E7 proteins of low- and high-risk human papillomaviruses share the ability to target the pRB family member p130 for degradation
Authors: Zhang Benyue , Chen Wei , Roman Ann
Published: December 2005

High-risk human papillomaviruses (HPVs) (e.g., HPV-16) cause anogenital and head and neck cancers, and low-risk HPVs (e.g., HPV-6) cause benign hyperproliferative disease. The E7 protein of HPV-16 binds all retinoblastoma tumor suppressor protein (pRB) family members with higher affinity than HPV-6E7. The HPV-16 E7 protein has been reported to target pRB family members for degradation and to immortalize cells. Here we tested the hypothesis that the low-risk E7 protein has an intrinsic ability to decrease expression of pRB family members. First, we introduced a high-affinity pRB-binding site into HPV-6 E7 (6E7G22D) and showed that, in human foreskin keratinocytes, HPV-6 E7G22D decreased the level of pRB protein but not pRB mRNA. Second, we analyzed the ability of wild-type HPV-6 E7 to destabilize the other pRB family members, p107 and p130. HPV-6 E7, like HPV-16 E7, decreased the level of p130 protein. This decrease was inhibited by MG132, a proteasome inhibitor. Binding of HPV-6 E7 to p130 was necessary but not sufficient to decrease the level of p130. Furthermore, the destabilization of p130 correlated with a decrease in the expression of involucrin, a differentiation marker. We suggest that the shared activity of HPV-16 E7 and HPV-6 E7 to destabilize p130 and decrease or delay differentiation may be related to the role of E7 in the HPV life cycle. The added ability of HPV-16 E7 to regulate pRB and p107 may be related to oncogenic activity.
Phosphorylation of the Chromosomal Passenger Protein Bir1 Is Required for Localization of Ndc10 to the Spindle during Anaphase and Full Spindle Elongation
Authors: Widlund Per O. , Lyssand John S. , Anderson Scott , Niessen Sherry , Yates John R. , Davis Trisha N.
Published: December 2005

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 μm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.
Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma Membrane
Authors: Alberts Philipp , Rudge Rachel , Irinopoulou Theano , Danglot Lydia , Gauthier-Rouvière Cécile , Galli Thierry
Published: December 2005

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.
The Association of Shiga-like Toxin with Detergent-resistant Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the Endoplasmic Reticulum for a Cytotoxic Effect
Authors: Smith Daniel C. , Sillence Daniel J. , Falguières Thomas , Jarvis Rosemary M. , Johannes Ludger , Lord J. Michael , Platt Frances M. , Roberts Lynne M.
Published: December 2005

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb3) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.
Sphingoid Base Is Required for Translation Initiation during Heat Stress inSaccharomyces cerevisiae
Authors: Meier Karsten D. , Deloche Olivier , Kajiwara Kentaro , Funato Kouichi , Riezman Howard
Published: December 2005

Sphingolipids are required for many cellular functions including response to heat shock. We analyzed the yeast lcb1-100 mutant, which is conditionally impaired in the first step of sphingolipid biosynthesis and shows a strong decrease in heat shock protein synthesis and viability. Transcription and nuclear export of heat shock protein mRNAs is not affected. However, lcb1-100 cells exhibited a strong decrease in protein synthesis caused by a defect in translation initiation under heat stress conditions. The essential lipid is sphingoid base, not ceramide or sphingoid base phosphates. Deletion of the eIF4E-binding protein Eap1p in lcb-100 cells restored translation of heat shock proteins and increased viability. The translation defect during heat stress in lcb1-100 was due at least partially to a reduced function of the sphingoid base-activated PKH1/2 protein kinases. In addition, depletion of the translation initiation factor eIF4G was observed in lcb1-100 cells and ubiquitin overexpression allowed partial recovery of translation after heat stress. Taken together, we have shown a requirement for sphingoid bases during the recovery from heat shock and suggest that this reflects a direct lipid-dependent signal to the cap-dependent translation initiation apparatus.
SMOKE‐FREE AIR LAWS, CIGARETTE PRICES, AND ADULT CIGARETTE DEMAND
Authors: TAURAS JOHN A.
Published: December 2005

This article examines the impact of cigarette prices and smoke‐free air laws on adult smoking. Probit methods and a generalized linear model with log‐link and Gaussian distribution are employed to model adult smoking propensity and intensity, respectively. After controlling for unobserved state‐level heterogeneity, which can influence both tobacco policy and smoking behavior, the estimates from this study imply that an inverse relationship exists between cigarette prices and both smoking prevalence and average cigarette consumption by adult smokers. The estimates also imply that more restrictive smoke‐free air laws decrease average smoking by adult smokers but have little impact on prevalence. (JEL I18)
Comparison of the aflR gene sequences of strains in Aspergillus section Flavi
Authors: Lee Chao-Zong , Liou Guey-Yuh , Yuan Gwo-Fang
Published: December 2005

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius and a few other species. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. Although Aspergillus oryzae and Aspergillus sojae, which are used in fermented foods and in ingredient manufacture, have no record of producing aflatoxin, they have been shown to possess an aflR gene. This study examined 34 strains of Aspergillus section Flavi. The aflR gene of 23 of these strains was successfully amplified and sequenced. No aflR PCR products were found in five A. sojae strains or six strains of A. oryzae. These PCR results suggested that the aflR gene is absent or significantly different in some A. sojae and A. oryzae strains. The sequenced aflR genes from the 23 positive strains had greater than 96·6 % similarity, which was particularly conserved in the zinc-finger DNA-binding domain. The aflR gene of A. sojae has two obvious characteristics: an extra CTCATG sequence fragment and a C to T transition that causes premature termination of AFLR protein synthesis. Differences between A. parasiticus/A. sojae and A. flavus/A. oryzae aflR genes were also identified. Some strains of A. flavus as well as A. flavus var. viridis, A. oryzae var. viridis and A. oryzae var. effuses have an A. oryzae-type aflR gene. For all strains with the A. oryzae-type aflR gene, there was no evidence of aflatoxin production. It is suggested that for safety reasons, the aflR gene could be examined to assess possible aflatoxin production by Aspergillus section Flavi strains.
Regulation of aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase in Lactobacillus plantarum
Authors: Cahyanto Muhammad N. , Kawasaki Hiroko , Nagashio Mariko , Fujiyama Kazuhito , Seki Tatsuji
Published: December 2005

The use of a lysine-overproducing strain ofLactobacillus plantarumin food or feed fermentations may lead to the production of lysine-rich products. The availability of functional genes and information on the regulation of lysine biosynthesis are required to develop a lysine-overproducing strain. The genome sequence ofL. plantarumrevealed putative lysine biosynthetic genes, some of which may produce isozymes. This study examined the functionality of the genes and the regulation of the first four enzymes of lysine biosynthesis, together with homoserine dehydrogenase, inL. plantarum. The genes were expressed inEscherichia coli, and the regulation of the enzymes was studied in cell extracts of both recombinantE. coliandL. plantarum. Among seven lysine biosynthetic genes studied (aspartokinase genes,thrA1andthrA2; aspartate semialdehyde dehydrogenase genes,asd1andasd2; dihydrodipicolinate synthase genes,dapA1anddapA2; and the dihydrodipicolinate reductase gene,dapB) plus two homoserine dehydrogenase genes (hom1andhom2), the products of six genes, i.e.thrA2,asd2,dapA1,dapB,hom1andhom2, showed obvious enzyme activitiesin vitro. The product of one of the homoserine dehydrogenase genes,hom1, exhibited both homoserine dehydrogenase and aspartokinase activities. However, the aspartokinase activity was mainly due to ThrA2 and was inhibited byl-lysine and repressed byl-threonine, and the homoserine dehydrogenase activity was mainly due to Hom2 and was inhibited byl-threonine. The aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase were not regulated by the end-products of the pathway.
Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism
Authors: Remonsellez Francisco , Orell Alvaro , Jerez Carlos A.
Published: December 2005

It has been postulated that inorganic polyphosphate (polyP) and transport of metal–phosphate complexes could participate in heavy metal tolerance in some bacteria. To study if such a system exists in archaea, the presence of polyP was determined by the electron energy loss spectroscopy (EELS) procedure and quantified by using specific enzymic methods inSulfolobus acidocaldarius,Sulfolobus metallicusandSulfolobus solfataricus. All three micro-organisms synthesized polyP during growth, but onlyS. metallicusgreatly accumulated polyP granules. The differences in the capacity to accumulate polyP between these archaea may reflect adaptive responses to their natural environment. Thus,S. metallicuscould grow in and tolerate up to 200 mM copper sulfate, with a concomitant decrease in its polyP levels with increasing copper concentrations. On the other hand,S. solfataricuscould not grow in or tolerate more than 1–5 mM copper sulfate, most likely due to its low levels of polyP. ShiftingS. metallicuscells to copper sulfate concentrations up to 100 mM led to a rapid increase in their exopolyphosphatase (PPX) activity which was concomitant in time with a decrease in their polyP levels and a stimulation of phosphate efflux. Furthermore, copper in the range of 10 μM greatly stimulated PPX activity in cell-free extracts fromS. metallicus. The results strongly suggest that a metal tolerance mechanism mediated through polyP is functional in members of the genusSulfolobus. This ability to accumulate and hydrolyse polyP may play an important role not only in the survival of these micro-organisms in sulfidic mineral environments containing high toxic metals concentrations, but also in their applications in biomining.
Increased pathology in lungs of mice after infection with an α-crystallin mutant of Mycobacterium tuberculosis: changes in cathepsin proteases and certain cytokines
Authors: Stewart Julie N. , Rivera Hilda N. , Karls Russell , Quinn Frederick D. , Roman Jesse , Rivera-Marrero Carlos A.
Published: December 2005

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. InMycobacterium tuberculosistheα-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of theacrgene inM. tuberculosisH37Rv (Δacrstrain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesisin vivowas unknown. This study demonstrated that infection of C57BL6 mice with Δacrresults in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or Δacrresults in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in Δacr-infected mice and was associated with clusters of macrophages within lung granulomas. Δacr-infected mice also showed high serum levels of TNF-α, IFN-γand G-CSF, suggesting that Acr may play a role in modulating the host response to infection.
PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract
Authors: Buckles Eric L. , Wang Xiaolin , Lockatell C. Virginia , Johnson David E. , Donnenberg Michael S.
Published: December 2005

The phoU gene is the last cistron in the pstSCAB–phoU operon and functions as a negative regulator of the Pho regulon. The authors previously identified a phoU mutant of extraintestinal pathogenic Escherichia coli strain CFT073 and demonstrated that this mutant was attenuated for survival in the murine model of ascending urinary tract infection. It is hypothesized that the PhoU protein might serve as a urovirulence factor by indirectly affecting the expression of virulence-related genes. In this study, the phoU mutant was further characterized and PhoU was confirmed as a virulence factor. Western blot analysis demonstrated that insertion of the transposon in the phoU gene disrupted the expression of PhoU. The phoU mutant had derepressed alkaline phosphatase activity under phosphate-excess and -limiting conditions. In single-challenge murine ascending urinary tract infection experiments, quantitative cultures of urine, bladder and kidney revealed no significant differences between the phoU mutant strain and the wild-type strain CFT073. However, in competitive colonization experiments, the phoU mutant strain was significantly out-competed by the wild-type strain in the kidneys and urine and recovered in lower amount in the bladder. Complementation of the phoU mutant with a plasmid containing the wild-type phoU gene restored the expression of PhoU and alkaline phosphate activity to wild-type levels and no significant difference in colonization was observed between the phoU mutant containing the complementing plasmid and wild-type in competitive colonization experiments. In human urine, the phoU mutant and wild-type grew comparably when inoculated independently, indicating that the attenuation observed was not due to a general growth defect. However, as observed in vivo, the wild-type out-competed the phoU mutant in competition growth experiments in human urine. These data indicate that PhoU contributes to efficient colonization of the murine urinary tract and add PhoU to a short list of confirmed urovirulence factors.
Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391
Authors: Girard Geneviève , van Rij E. Tjeerd , Lugtenberg Ben J. J. , Bloemberg Guido V.
Published: December 2005

Production of the secondary metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis PCL1391 is crucial for biocontrol activity against the phytopathogen Fusarium oxysporum f. sp. radicis lycopersici on tomato. Regulation of PCN production involves the two-component signalling system GacS/GacA, the quorum-sensing system PhzI/PhzR and the regulator PsrA. This paper reports that a functional rpoS is required for optimal PCN and N-hexanoyl-l-homoserine lactone (C6-HSL) production. Constitutive expression of rpoS is able to complement partially the defect of a psrA mutant for PCN and N-acylhomoserine lactone production. Western blotting shows that rpoS is regulated by gacS. Altogether, these results suggest the existence of a cascade consisting of gacS/gacA upstream of psrA and rpoS, which influence expression of phzI/phzR. Overproduction of phzR complements the effects on PCN and C6-HSL production of all mutations tested in the regulatory cascade, which shows that a functional quorum-sensing system is essential and sufficient for PCN synthesis. In addition, the relative amounts of PCN, phenazine-1-carboxylic acid and C6-HSL produced by rpoS and psrA mutants harbouring a constitutively expressed phzR indicate an even more complex network of interactions, probably involving other genes. Preliminary microarray analyses of the transcriptomics of the rpoS and psrA mutants support the model of regulation described in this study and allow identification of new genes that might be involved in secondary metabolism.
Molecular analysis of the glucose-specific phosphoenolpyruvate : sugar phosphotransferase system from Lactobacillus casei and its links with the control of sugar metabolism
Authors: Yebra María J. , Monedero Vicente , Zúñiga Manuel , Deutscher Josef , Pérez-Martínez Gaspar
Published: December 2005

Lactobacillus caseitransports glucose preferentially by a mannose-class phosphoenolpyruvate : sugar phosphotransferase system (PTS). The genomic analysis ofL. caseiallowed the authors to find a gene cluster (manLMNO) encoding the IIAB (manL), IIC (manM) and IID (manN) proteins of a mannose-class PTS, and a putative 121 aa protein of unknown function (encoded bymanO), homologues of which are also present inmanclusters that encode glucose/mannose transporters in other Gram-positive bacteria. TheL. casei manoperon is constitutively expressed into amanLMNOmessenger, but an additionalmanOtranscript was also detected. Upstream of themanoperon, two genes (upsRandupsA) were found which encode proteins resembling a transcriptional regulator and a membrane protein, respectively. Disruption of eitherupsRorupsAdid not affectmanLMNOtranscription, and had no effect on glucose uptake. Cells carrying amanOdeletion transported glucose at a rate similar to that of the wild-type strain. By contrast, amanMdisruption resulted in cells unable to transport glucose by the PTS, thus confirming the functional role of themangenes. In addition, themanMmutant exhibited neither inducer exclusion of maltose nor glucose repression. This result confirms the need for glucose transport through the PTS to trigger these regulatory processes inL. casei.
Characterization of the theta-type plasmid pCD3.4 from Carnobacterium divergens, and modulation of its host range by RepA mutation
Authors: van Belkum Marco J. , Stiles Michael E.
Published: December 2005

The complete nucleotide sequence of the 3475 bp plasmid pCD3.4 from Carnobacterium divergens LV13, which encodes the bacteriocin divergicin A, was determined. Nucleotide sequence, deletion and complementation analyses revealed the presence of a trans-acting replication protein, RepA, and DNA sequences involved in plasmid replication and copy-number control. The DNA region preceding the repA gene probably contains the origin of replication. This sequence includes four and a half direct repeats (iterons) of 22 bp, to which RepA is thought to bind, and an AT-rich region containing a 12 bp repeat, at which initiation of DNA might occur. Further upstream of this sequence resides a fifth iteron required for optimal plasmid replication. The RepA protein shows homology to replication proteins of the pUCL287 subfamily of theta-type replicons. Two ORFs were found downstream of the repA gene that could be deleted without affecting replication and stability of the plasmid. pCD3.4 has a narrow host range, and could only be maintained in Carnobacterium spp.; however, a mutant of the plasmid was obtained that enabled the pCD3.4 replicon to replicate in Enterococcus faecium, but not in Carnobacterium spp. The mutation was located in the C-terminal region of the RepA protein, changing a proline into a serine. This is believed to be the first example of such plasmid-host-range modulation in Gram-positive bacteria.
Lipoprotein computational prediction in spirochaetal genomes
Authors: Setubal João C. , Reis Marcelo , Matsunaga James , Haake David A.
Published: December 2005

Lipoproteins are of great interest in understanding the molecular pathogenesis of spirochaetes. Because spirochaete lipobox sequences exhibit more plasticity than those of other bacteria, application of existing prediction algorithms to emerging sequence data has been problematic. In this paper a novel lipoprotein prediction algorithm is described, designated SpLip, constructed as a hybrid of a lipobox weight matrix approach supplemented by a set of lipoprotein signal peptide rules allowing for conservative amino acid substitutions. Both the weight matrix and the rules are based on a training set of 28 experimentally verified spirochaetal lipoproteins. The performance of the SpLip algorithm was compared to that of the hidden Markov model-based LipoP program and the rules-based algorithm Psort for all predicted protein-coding genes of Leptospira interrogans sv. Copenhageni, L. interrogans sv. Lai, Borrelia burgdorferi, Borrelia garinii, Treponema pallidum and Treponema denticola. Psort sensitivity (13–35 %) was considerably less than that of SpLip (93–100 %) or LipoP (50–84 %) due in part to the requirement of Psort for Ala or Gly at the −1 position, a rule based on E. coli lipoproteins. The percentage of false-positive lipoprotein predictions by the LipoP algorithm (8–30 %) was greater than that of SpLip (0–1 %) or Psort (4–27 %), due in part to the lack of rules in LipoP excluding unprecedented amino acids such as Lys and Arg in the −1 position. This analysis revealed a higher number of predicted spirochaetal lipoproteins than was previously known. The improved performance of the SpLip algorithm provides a more accurate prediction of the complete lipoprotein repertoire of spirochaetes. The hybrid approach of supplementing weight matrix scoring with rules based on knowledge of protein secretion biochemistry may be a general strategy for development of improved prediction algorithms.
Genetic and functional analyses of the lgtH gene, a member of the β-1,4-galactosyltransferase gene family in the genus Neisseria
Authors: Zhu Peixuan , Boykins Robert A. , Tsai Chao-Ming
Published: December 2005

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Three galactosyltransferase genes, lgtB, lgtE and lgtH, responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five Neisseria species. The function of lgtH in Neisseria meningitidis 6275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that lgtH encodes a β-1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the α chain is the acceptor site in the biosynthesis of Neisseria LOS. To understand the sequence diversity and relationships of lgtB, lgtE and lgtH, the entire lgt-1 locus was further sequenced in three N. meningitidis strains and three commensal Neisseria strains, and compared with the previously reported lgt genes from Neisseria species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 lgtB, 12 lgtE and 14 lgtH sequences revealed a distinct evolutionary history of these genes in Neisseria. For example, the splits graph of lgtE displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of lgtB and lgtH displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.
Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport
Authors: Delgado María J. , Tresierra-Ayala Alvaro , Talbi Chouhra , Bedmar Eulogio J.
Published: December 2005

A modABC gene cluster that encodes an ABC-type, high-affinity molybdate transporter from Bradyrhizobium japonicum has been isolated and characterized. B. japonicum modA and modB mutant strains were unable to grow aerobically or anaerobically with nitrate as nitrogen source or as respiratory substrate, respectively, and lacked nitrate reductase activity. The nitrogen-fixing ability of the mod mutants in symbiotic association with soybean plants grown in a Mo-deficient mineral solution was severely impaired. Addition of molybdate to the bacterial growth medium or to the plant mineral solution fully restored the wild-type phenotype. Because the amount of molybdate required for suppression of the mutant phenotype either under free-living or under symbiotic conditions was dependent on sulphate concentration, it is likely that a sulphate transporter is also involved in Mo uptake in B. japonicum. The promoter region of the modABC genes has been characterized by primer extension. Reverse transcription and expression of a transcriptional fusion, PmodA–lacZ, was detected only in a B. japonicum modA mutant grown in a medium without molybdate supplementation. These findings indicate that transcription of the B. japonicum modABC genes is repressed by molybdate.
Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals
Authors: Miller William G. , Englen Mark D. , Kathariou Sophia , Wesley Irene V. , Wang Guilin , Pittenger-Alley Lauren , Siletz Robin M. , Muraoka Wayne , Fedorka-Cray Paula J. , Mandrell Robert E.
Published: December 2005

Campylobacter coli is a food-borne pathogen associated increasingly with human gastroenteritis. C. coli has a high prevalence in swine, but is isolated also from cattle and poultry. Multilocus sequence typing (MLST) systems have been developed to differentiate C. coli strains. Although substantial allelic diversity was identified across all seven C. coli MLST loci, no correlations were made in two previous studies between allele or sequence type (ST) and the source of the organism. However, this may be due to either the relatively small number or the low diversity of C. coli strains used to validate both MLST studies. This study describes the typing of 488 C. coli strains from 4 different food animal sources (cattle, chickens, swine and turkeys), collected at different times over a 6 year period from different USA geographical locations. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52/63 (83 %) strains possessed a single ST (ST-1068). A subpopulation of C. coli strains, collected primarily from turkeys, was identified, containing both C. coli- and Campylobacter jejuni-associated MLST alleles, specifically the C. jejuni allele aspA103. The majority of STs and alleles were host associated, i.e. found primarily in strains from a single food-animal source. Only 12/149 (8 %) STs were found in multiple sources. Additionally, the majority (34/46, 74 %) of major (n>5) alleles were more prevalent in certain hosts (swine, poultry). The presence of host-associated C. coli MLST alleles could lead potentially to more efficient source tracking in this species, especially in the trace-back of both sporadic and outbreak human clinical C. coli strains to animal sources.
Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals
Authors: Shaver Ciara M. , Hauser Alan R.
Published: December 2005

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins ofPseudomonas aeruginosaresult in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination.In vitrocytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.
Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore
Authors: Maunsell Bláithín , Adams Claire , O'Gara Fergal
Published: December 2005

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.
Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH
Authors: Barriuso-Iglesias Mónica , Barreiro Carlos , Flechoso Fabio , Martín Juan F.
Published: December 2005

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7·0–9·0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9·0. Growth still occurred at pH 9·5 but at a reduced rate. The expression of the pH-regulated F0F1ATPase operon (containing the eight genesatpBEFHAGDC) was induced at alkaline pH. A 7·5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9·0. The same occurred with a 1·2 kb transcript corresponding to theatpBgene. RT-PCR studies confirmed the alkaline pH induction of the F0F1operon and the existence of theatpIgene. TheatpIgene, located upstream of the F0F1operon, was expressed at a lower level than the polycistronic 7·5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0F1operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional inC. glutamicumandEscherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetativeσfactor ofC. glutamicum, whereas the −35 and −10 boxes of P-atp2 fitted the consensus sequence forσH-recognizedMycobacterium tuberculosispromoters CC/GGGA/GAC 17–22 ntC/GGTTC/G, known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0F1operon is highly expressed at alkaline pH, probably using aσHRNA polymerase.