bluuebunny/crossref_metadata_2025 huggingface dataset:
Endoplasmic reticulum stress caused by aggregate‐prone proteins containing homopolymeric amino acids
Authors: Uchio Naohiro , Oma Yoko , Toriumi Kazuya , Sasagawa Noboru , Tanida Isei , Fujita Eriko , Kouroku Yoriko , Kuroda Reiko , Momoi Takashi , Ishiura Shoichi
Published: October 2007

Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate‐prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well‐known aggregate‐prone polyglutamine‐containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate‐prone proteins such as polyglutamine.
The hydroxyproline motif of male sex peptide elicits the innate immune response in Drosophila females
Authors: Domanitskaya Elena V. , Liu Huanfa , Chen Shanjun , Kubli Eric
Published: October 2007

Seminal fluid elicits a variety of physiological and behavioral changes in insect females. In Drosophila melanogaster females, sex peptide (SP) is the major seminal agent eliciting oviposition and reduction of receptivity. But SP also has many other effects; for example, it stimulates food intake, egg production, ovulation, juvenile hormone production and antimicrobial peptide synthesis. Thus, SP very probably has several receptors. To identify putative targets and signaling cascades, we studied the genome‐wide regulation of genes by microarray analysis of RNA isolated from females after mating with wild‐type males or males lacking SP, respectively. In addition, we studied the effects of SP on the proteome of females. Sex peptide regulates gene activity differentially in the head and in the abdomen. Genes coding for unspecific antimicrobial peptides are specifically transcribed in the abdomen, e.g. the antimicrobial peptide drosocin in epithelial tissues of the female genital tract (oviduct and calyx). Hence, SP elicits a systemic [Peng J, Zipperlen P & Kubli E (2005) Curr Biol15, 1690–1694] and an epithelial immune response. Ectopic expression of SP in the fat body of transgenic virgin females (with subsequent secretion into the hemolymph) does not elicit drosocin synthesis in the genital tract. Thus, the receptors for the stimulation of the systemic and the epithelial responses by SP are compartmentalized. The hydroxyproline (P*) motif of SP, P*TKFP*IP*SP*NP*, is identified as a novel elicitor of the innate immune response. We suggest that SP acts by chemical mimicry of sugar components of the bacterial cell wall. Thus, SP may induce the immune system via pattern recognition receptors.
Analysis of the CK2‐dependent phosphorylation of serine 13 in Cdc37 using a phospho‐specific antibody and phospho‐affinity gel electrophoresis
Authors: Miyata Yoshihiko , Nishida Eisuke
Published: October 2007

The CK2‐dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase‐specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065–4074]. Here we describe a new phospho‐specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg2+ and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506‐binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v‐Src, and Raf1, contained the CK2‐phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor‐induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho‐affinity gel electrophoresis. Our analyses demonstrated that the CK2‐dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2‐dependent Cdc37 phosphorylation and the usefulness of phospho‐affinity gel electrophoresis in protein phosphorylation analysis.
Saccharomyces cerevisiae Cox18 complements the essential Sec‐independent function of Escherichia coli YidC
Authors: van Bloois Edwin , Koningstein Gregory , Bauerschmitt Heike , Herrmann Johannes M. , Luirink Joen
Published: October 2007

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec‐translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec‐independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.
Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis
Authors: Hennig Anna , Bonfig Katharina , Roitsch Thomas , Warzecha Heribert
Published: October 2007

Bacterial lipoproteins play crucial roles in host–pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N‐terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten‐fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N‐terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration‐dependent interference with essential functions of chloroplastic membranes required for primary metabolism.
Chronic ulcers, calcification and calcified fibrous tumours: phenotypic manifestations of a congenital disorder of heterotopic ossification
Authors: Kupitz Susanne , Enoch Stuart , Harding Keith G
Published: October 2007

Calcification is a rarely reported cause for chronic, cutaneous ulceration. Although dystrophic calcification occurs in chronic ulcers, idiopathic calcification and ossification leading to recurrent ulcerations is seldom reported. This report illustrates a challenging case with various non healing wounds, calcification/ossification and calcified fibrous tumours. A 56‐year‐old woman presented with chronic, painful wounds and calcified deposits in her feet, hands and abdomen, some dating back to childhood. The surgeries she had had for various unrelated conditions healed with hard, calcified deposits, which later ulcerated. Skeletal radiological imaging revealed multiple soft‐tissue deposits, with calcification and areas of ossification, along with gross distortion of the bony architecture of her feet. All biochemical investigations were normal apart from raised serum alkaline phosphatase. The management has encompassed a combination of conservative and surgical measures (culminating in a left below‐knee amputation) with varying degrees of success. Having ruled out other causes of subcutaneous calcification, three congenital disorders of heterotopic ossification fit this patient’s presentation: Albright hereditary osteodystrophy (AHO), fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia (POH). Although AHO and FOP are possibilities, POH is most likely. In addition to describing the diverse phenotypic manifestations of this disorder, this report discusses the diagnostic dilemmas, difficulties in optimising the management plan and issues relating to health‐related quality of life in this patient.
Effects of pH‐neutral, super‐oxidised solution on human dermal fibroblasts in vitro
Authors: González‐Espinosa Diana , Pérez‐Romano Leafar , Guzmán‐Soriano Beatriz , Arias Edmundo , Bongiovanni Cheryl M , Gutiérrez Andrés A
Published: October 2007

For the past 20 years, super‐oxidised solutions (SOSs) have been shown to be potent antimicrobials and disinfectants via oxidative damage. However, the potential toxicity of SOSs on eukaryotic cells has not been documented in vitro. This is relevant because oxygen and chlorine reactive species may possibly induce ageing and irreversible cellular dysfunctions that eventually produce cell death. The present study investigates the cytotoxicity and oxidative stress induced by a novel, pH‐neutral SOS (i.e. Microcyn™, MCN) on young, primary diploid – human dermal fibroblast (HDF) cultures. For this purpose, hydrogen peroxide (HP) was used as a positive control of oxidative damage. When these solutions were used at concentrations indicated for wound care (i.e. undiluted MCN or 880 mM HP), HP was significantly more toxic than MCN. After 5 and 30 minutes of exposure, cell viability was 38% and 5%, respectively, in 880 mM HP‐treated cells versus 75% and 70% in MCN‐treated populations, respectively. HP induced both apoptosis and necrosis, whereas MCN induced only necrosis. Genotoxic and ageing studies were then conducted at sublethal HP concentrations as previously reported in the literature. Cellular DNA and RNA were partially degraded only in HDFs exposed to 500 μM HP for 30 minutes but not in those exposed to undiluted MCN. At this same concentration, HP induced the formation of 8‐hydroxy‐2′deoxyguanosine adducts in HDFs but this effect was neither observed in control‐ nor observed in MCN‐treated cells. HDFs were further exposed to 5 μM HP or 10% MCN for 1 month. The expression of senescence‐associated‐β‐galactosidase was only significantly elevated in cells chronically exposed to 5 μM HP. Altogether, these results show that MCN is significantly less cytotoxic than antiseptic HP concentrations (i.e. 880 mM) and that, in vitro, it does not induce genotoxicity or accelerated ageing.
Changes in plant–soil hydraulic pressure gradients of soybean in response to soil drying
Authors: Sinclair T.R. , Zwieniecki M.A. , Holbrook N.M.
Published: October 2007

Plant gas‐exchange response to drying soil in many instances tracks a common pattern when expressed as a function of fraction of transpirable soil water (FTSW). There is little decrease in gas exchange until FTSW decreases to a value in the range of about 0.3–0.45, then with further drying gas exchange declines approximately linearly. This unique pattern is hypothesised to reflect mainly changes in the water potential gradient between bulk soil and plant. The primary objective was to directly document the basis of this response by measuring the hydrostatic pressure gradient required in the soil to maintain leaf xylem at zero potential with decreasing FTSW. Pots in which soybean (Glycine max) plants were grown were placed in a pressure chamber and the pressure adjusted to maintain zero water potential in a leaf petiolule. These results showed a small, relatively constant hydrostatic pressure had to be applied to the soil to maintain zero leaf xylem water potential until FTSW decreased to approximately 0.3–0.45. Thereafter, the required hydrostatic pressure gradient increased as FTSW continued to decrease. Hydraulic conductance was calculated to be relatively stable early in the drying cycle, and then decrease as the soil dried to comparatively high FTSW of 0.5–0.7.
‘Something distinctly not of this character’: how Knightian uncertainty is relevant to corporate governance
Authors: Belcher Alice
Published: October 2007

Corporate governance best practice now includes the requirement for a board to ensure that the system of internal control is effective in managing risks in the manner which it has approved. The Turnbull Guidance on this matter is ‘based on the adoption by a company's board of a risk-based approach to establishing a sound system of internal control and reviewing its effectiveness’. This involves the identification and prioritising of risks and embedding the risk management approach in the culture and processes of the business. This article explores the use of risk assessment in the law, particularly as it applies to the activities of companies. It is suggested that a concentration on, and perhaps a spurious quantification of, risk could mask the difficulty of making board-level decisions under conditions of true or Knightian uncertainty. Knight maintained that true uncertainty was something distinctly not of the character of risk. It occurs when ‘. . . there is no possibility of forming in any way groups of instances of sufficient homogeneity to make possible a quantitative determination of true probability. Business decisions, for example, deal with situations which are far too unique, generally speaking, for any sort of statistical tabulation to have any value for guidance. The concept of objectively measurable probability or chance is simply inapplicable’.
Prognostic implications of a biphasic waveform for APTT analysis in a district general hospital
Authors: MAIR G. , DUNHILL S. , TIPLADY C.
Published: October 2007

Activated partial thromboplastin time analysis creates waveforms of light transmittance against time. An abnormal biphasic waveform has been linked to morbidity and mortality. This study aims to quantify this link for patients in a district general hospital and determine the prognostic implications of the biphasic waveform. Data were collected over a 5‐month period and analysed retrospectively to ascertain rates of infection, disseminated intravascular coagulation (DIC) and death. This was followed by a one month prospective study to assess prognostic implications. Rates of infection, DIC and death were high in this study population. The biphasic waveform often pre‐empts these outcomes and is shown to be useful in predicting a poor prognosis.
New REE and Trace Element Data on Two Kimberlitic Reference Materials by ICP‐MS
Authors: Roy Parijat , Balaram Vysetti , Kumar Anil , Satyanarayanan Manavalan , Gnaneshwar Rao Thota
Published: October 2007

Data on thirty‐four minor and trace elements including all rare earth elements (REE) are reported for two kimberlitic international reference materials (SARM‐39, MINTEK, RSA and MY‐4, IGEM, Russia) by inductively coupled plasma‐mass spectrometry (ICP‐MS), some of them for the first time. Four digestion techniques (open acid, closed vessel acid, microwave and lithium metaborate fusion digestion) were used for the decomposition of samples for analysis by ICP‐MS. Three other reference materials (USGS BHVO‐1, CRPG BR‐1 and ANRT UB‐N) were analysed simultaneously using the same analytical methodology to assess the precision and accuracy of the determinations. The data obtained in this study compare well with working values wherever such values are available for comparison. Though open acid digestion was found to be very rapid, effective and convenient for the determination of several trace elements in kimberlitic samples, recoveries for heavy rare earth elements (HREE) were lower than the respective recoveries obtained by the other decomposition techniques used. The precision obtained was better than ± 6% RSD in the majority of cases with comparable accuracy. Chondrite‐normalised plots of each RM for all the digestion techniques were smooth. The new data reported on the two kimberlitic reference materials make these samples useful for future geochemical studies of kimberlitic rocks.
High‐Performance Liquid and Ion Chromatography: Separation and Quantification Analytical Techniques for Rare Earth Elements
Authors: Verma Surendra P. , Santoyo Edgar
Published: October 2007

High‐performance liquid chromatography (HPLC) and ion chromatography (HPIC) have been widely used as cheap, relatively rapid, precise and accurate separation and quantification analytical techniques for the rare earth elements (REE). In this “Back to Basics” review, a general description of the basic components of instrumentation and the most common detectors is presented, with special attention to separation conditions and chromatographic quality parameters for the REEs. Also included is a brief survey of the literature on the use of HPLC and HPIC in the determination of REEs in synthetic solutions as well as other matrices such as nuclear fuels and geochemical reference materials. For a proper evaluation of HPLC and HPIC applications, and use of these techniques by other researchers it is recommended that parameters such as the amount and uncertainty estimates of an element injected, eluents and post‐column reagents and information on linear or complex gradients and detector system set‐up, should always be reported or relevant literature references provided. Finally, a brief account is also presented on state‐of‐the‐art liquid chromatography known as ultra high‐ or extremely high‐performance liquid chromatography, which has high potential for a diversity of applications in all scientific and engineering fields including the Earth Sciences.
Determination of Total Organic Carbon Content and δ13C in Carbonate‐Rich Detrital Sediments
Authors: Galy Valier , Bouchez Julien , France‐Lanord Christian
Published: October 2007

The determination of total organic carbon content and composition in detrital sediments requires careful removal of their carbonate minerals. In detrital sediments containing large amounts of carbonates, including dolomite, this can only be achieved by liquid acid leaching that may solubilise a significant proportion of the organic carbon. For a set of detrital sediments from the Himalayan system and the Amazon River as well as five geological reference materials, we determined the proportion of organic carbon (Corg) solubilised during acid leaching. This proportion is significant for all analysed sediments and generally tends to increase with the organic carbon content. Compared to other types of sediments analysed, clay fractions extracted from river sediments and bed sediments with very low organic carbon content have high and low proportions of acid soluble Corg respectively. In Himalayan and Amazon river sediments, the proportion of Corg solubilised during acid leaching was relatively constant with average values of 14 and 19 % respectively. Thus, it is possible to correct the Corg content for the dissolved organic carbon content measured after decarbonation. Data presented here show that Corg dissolved during liquid acid leaching must be taken into account. After careful calibration, the method presented here should, therefore, be applied to any carbonate‐rich detrital sediment.
Methodology for Rare Earth Element Determinations of Uranium Oxides by Ion Microprobe
Authors: Bonhoure Jessica , Kister Philippe , Cuney Michel , Deloule Etienne
Published: October 2007

A methodology for the determination of the rare earth elements in uranium oxides by ion microprobe has been set up on a Cameca ims‐3f instrument. An uranium oxide reference material from a syn‐metamorphic uranium deposit related to albitisation has also been developed for this type of analysis. Applications of the methodology are presented for a series of uranium oxides selected from some major uranium deposit types: from the world's highest grade unconformity‐related uranium deposit from the Athabasca Basin (Saskatchewan, Canada; the Shea Creek and the McArthur River examples), a perigranitic vein‐type deposit (Pen Ar Ran, Vendée, France) and a volcanic caldera‐related deposit (Streltsovkoye, Transbaikalia, Russia). Each type of uranium deposit appears to have a specific REE signature. All REE patterns from the Shea Creek and the McArthur deposits are characterised by bell‐shaped patterns centred on Tb‐Dy and similar to those already published for uranium oxides from unconformity‐related deposits from Australia. Such bell‐shaped REE patterns centred on Tb‐Dy may therefore be considered as a typical signature of uranium oxides from Mesoproterozoic unconformity‐related deposits. A smoother bell shape pattern centred on Eu characterises the syn‐metamorphic albitisation related deposit of Mistamisk selected for the reference material. The REE patterns from the Pen Ar Ran deposit show a fractionation from LREE to HREE with anomalously high abundances of Sm, Eu and Gd with respect to the other REEs, similar to the REE patterns of uranium oxides from the volcanic‐related deposits of Streltsovkoye.
Quantitative Distributions of
Epsilonproteobacteria
and a
Sulfurimonas
Subgroup in Pelagic Redoxclines of the Central Baltic Sea
Authors: Grote Jana , Labrenz Matthias , Pfeiffer Birgit , Jost Günter , Jürgens Klaus
Published: October 2007

Members of the class
Epsilonproteobacteria
are known to be of major importance in biogeochemical processes at oxic-anoxic interfaces. In pelagic redoxclines of the central Baltic Sea, an uncultured epsilonproteobacterium related to
Sulfurimonas denitrificans
was proposed to play a key role in chemolithotrophic denitrification (I. Brettar, M. Labrenz, S. Flavier, J. Bötel, H. Kuosa, R. Christen, and M. G. Höfle, Appl. Environ. Microbiol. 72:1364-1372, 2006). In order to determine the abundance, activity, and vertical distribution of this bacterium in high-resolution profiles, 16S rRNA cloning and catalyzed reporter deposition and fluorescence in situ hybridization (CARD-FISH) and quantitative PCR measurements were carried out. The results showed that 21% of the derived clone sequences, which in the present study were grouped together under the name GD17, had >99% similarity to the uncultured epsilonproteobacterium. A specific gene probe against GD17 (S-*-Sul-0090-a-A-18) was developed and used for enumeration by CARD-FISH. In different pelagic redoxclines sampled during August 2003, May 2005, and February 2006, GD17 cells were always detected from the lower oxic area to the sulfidic area. Maximal abundance was detected around the chemocline, where sulfide and nitrate concentrations were close to the detection limit. The highest GD17 numbers (2 × 10
5
cells ml
−1
), representing up to 15% of the total bacteria, were comparable to those reported for
Epsilonproteobacteria
in pelagic redoxclines of the Black Sea and the Cariaco Trench (X. Lin, S. G. Wakeham, I. F. Putnam, Y. M. Astor, M. I. Scranton, A. Y. Chistoserdov, and G. T. Taylor, Appl. Environ. Microbiol. 72:2679-2690, 2006). However, in the Baltic Sea redoxclines,
Epsilonproteobacteria
consisted nearly entirely of cells belonging to the distinct GD17 group. This suggested that GD17 was the best-adapted epsilonproteobacterium within this ecological niche.
Biosurfactant Production and Surface Translocation Are Regulated by PlcR in
Bacillus cereus
ATCC 14579 under Low-Nutrient Conditions
Authors: Hsueh Yi-Huang , Somers Eileen B. , Lereclus Didier , Ghelardi Emilia , Wong Amy C. Lee
Published: October 2007

Bacillus cereus
ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The
B. cereus
ATCC 14579 Δ
plcR
mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K
2
HPO
4
, 3.0 g KH
2
PO
4
, 0.1 g MgSO
4
·7H
2
O, 0.1 g (NH
4
)
2
SO
4
, 0.01 g CaCl
2
, 0.001 g FeSO
4
, 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by
Bacillus subtilis
) or biosurfactant purified from the Δ
plcR
mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and Δ
plcR
mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the Δ
plcR
mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the Δ
plcR
mutant was a lipopeptide and had a mass of 1,278.1722 (
m
/
z
). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.
Analysis of Nitrogen-Fixing Members of the ε Subclass of
Proteobacteria
in Salt Marsh Sediments
Authors: Welsh Allana , Burke David J. , Hahn Dittmar
Published: October 2007

Based on phylogenetic analysis of clones retrieved from two
nifH
gene clone libraries that were created using cDNA from suboxic sediment samples obtained from areas densely vegetated with the high-salt marsh plant
Spartina patens
, a primer set was designed to target nitrogen-fixing bacteria with sequence similarities to members of the ε subclass of
Proteobacteria
. Nested PCR, denaturing gel electrophoresis, and subsequent sequence analysis of reamplified fragments confirmed the specificity of the primer set by retrieving
nifH
sequences of only putative members of the ε subclass of
Proteobacteria
, all of which were characterized by a highly divergent 27- or 36-bp insertion in both DNA and cDNA.
Novel
Cryptosporidium
Genotype in Wild Australian Mice (
Mus domesticus
)
Authors: Foo Colin , Farrell Julianne , Boxell Annika , Robertson Ian , Ryan Una M.
Published: October 2007

A total of 250 mouse fecal specimens collected from crop farms in Queensland, Australia, were screened for the presence of
Cryptosporidium
spp. using PCR. Of these, 19 positives were detected and characterized at a number of loci, including the 18S rRNA gene, the acetyl coenzyme A gene, and the actin gene. Sequence and phylogenetic analyses identified two genotypes: mouse genotype I and a novel genotype (mouse genotype II), which is likely to be a valid species.
Cryptosporidium parvum
, which is zoonotic, was not detected. The results of the study indicate that wild Australian mice that are not in close contact with livestock are probably not an important reservoir of
Cryptosporidium
infection for humans and other animals.
Metagenomic Characterization of Chesapeake Bay Virioplankton
Authors: Bench Shellie R. , Hanson Thomas E. , Williamson Kurt E. , Ghosh Dhritiman , Radosovich Mark , Wang Kui , Wommack K. Eric
Published: October 2007

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of
Siphophage
homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the
psbA
gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of
psbA
homologous sequences to total cyanophage sequences in the metagenome indicated that the
psbA
gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of
psbD
homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by
Podoviridae
.
Chromosomal and Symbiotic Relationships of Rhizobia Nodulating
Medicago truncatula
and
M. laciniata
Authors: van Berkum Peter , Badri Yazid , Elia Patrick , Aouani Mohammed Elarbi , Eardly Bertrand D.
Published: October 2007

Multilocus sequence typing (MLST) is a sequence-based method used to characterize bacterial genomes. This method was used to examine the genetic structure of
Medicago
-nodulating rhizobia at the Amra site, which is located in an arid region of Tunisia. Here the annual medics
Medicago laciniata
and
M. truncatula
are part of the natural flora. The goal of this study was to identify whether distinct chromosomal groups of rhizobia nodulate
M. laciniata
because of its restricted requirement for specific rhizobia. The MLST analysis involved determination of sequence variation in 10 chromosomal loci of 74 isolates each of
M. laciniata
and
M. truncatula. M. truncatula
was used as a control trap host, because unlike
M. laciniata
, it has relatively unrestricted rhizobial requirements. Allelic diversity among the plasmid
nodC
alleles in the isolates was also determined. The 148 isolates were placed into 26 chromosomal sequence types (STs), only 3 of which had been identified previously. The rhizobia of
M. laciniata
were shown to be part of the general
Medicago
-nodulating population in the soil because 99.95% of the isolates had chromosomal genotypes similar to those recovered from
M. truncatula
. However, the isolates recovered from
M. laciniata
were less diverse than those recovered from
M. truncatula
, and they also harbored an unusual
nodC
allele. This could perhaps be best explained by horizontal transfer of the different
nodC
alleles among members of the
Medicago
-nodulating rhizobial population at the field site. Evidence indicating a history of lateral transfer of rhizobial symbiotic genes across distinct chromosomal backgrounds is provided.
Expanding the
Helicobacter pylori
Genetic Toolbox: Modification of an Endogenous Plasmid for Use as a Transcriptional Reporter and Complementation Vector
Authors: Carpenter Beth M. , McDaniel Timothy K. , Whitmire Jeannette M. , Gancz Hanan , Guidotti Silvia , Censini Stefano , Merrell D. Scott
Published: October 2007

Helicobacter pylori
is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous
H. pylori
plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an
Escherichia coli
origin of replication, a kanamycin resistance cassette, a promoterless
gfpmut3
gene, and a functional multiple cloning site to form pTM117. The promoters of
amiE
and
pfr
, two well-characterized Fur-regulated promoters, were fused to the promoterless
gfpmut3
, and green fluorescent protein (GFP) expression of the fusions in wild-type and Δ
fur
strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing
amiE
,
pfr
, and
fur
expression in wild-type and Δ
fur
strains carrying a wild-type copy of
fur
on the plasmid. Proper regulation of these genes was restored in the Δ
fur
background under high- and low-iron conditions, signifying complementation of both iron-bound and
apo
Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of
H. pylori
.
The
Listeria monocytogenes
Homolog of the
Escherichia coli era
Gene Is Involved in Adhesion to Inert Surfaces
Authors: Auvray Frédéric , Chassaing Danielle , Duprat Cécile , Carpentier Brigitte
Published: October 2007

Two transposon-insertional mutants of
Listeria monocytogenes
showing smaller viable surface-attached cell populations after disinfection with
N
,
N
-didecyl-
N
,
N
-dimethylammonium chloride were identified. In both mutants, transposon Tn
917-lac
was found to be inserted into the same gene, lmo1462, which is homologous to the essential
Escherichia coli era
gene. Both
L. monocytogenes
lmo1462-disrupted mutants displayed lower growth rates, as was also shown for several
E. coli era
mutants, and the lmo1462 gene was able to complement the growth defect of an
E. coli era
mutant. We showed that the disruption of lmo1462 decreased the ability of
L. monocytogenes
cells to adhere to stainless steel. Our results suggest that this
era
-like gene is involved in adhesion and contributes to the presence of
L. monocytogenes
on surfaces.
Effect of CO
2
on Colony Development by
Bifidobacterium
Species
Authors: Kawasaki Shinji , Nagasaku Masahiro , Mimura Tsuyoshi , Katashima Hitomi , Ijyuin Susumu , Satoh Takumi , Niimura Youichi
Published: October 2007

This report investigates the requirement for CO
2
for colony formation by
Bifidobacterium
species in both anoxic and oxic environments. All tested
Bifidobacterium
species exhibited difficulty in developing colonies in an atmosphere of 100% N
2
but developed well when 1% CO
2
was present. In the presence of CO
2
, the oxygen tolerance of the tested species was not improved. In the absence of CO
2
, only
B. boum
, a microaerophilic species, could develop colonies under an N
2
-based 5% O
2
atmosphere, indicating that while CO
2
is not an essential factor for colony development, both CO
2
and O
2
have stimulatory effects on
B. boum
colony development.
Detection of Bacteriophage-Infected Cells of
Lactococcus lactis
by Using Flow Cytometry
Authors: Michelsen Ole , Cuesta-Dominguez Álvaro , Albrechtsen Bjarne , Jensen Peter Ruhdal
Published: October 2007

Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in
Lactococcus lactis
by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret this change as a consequence of a cease in cell growth, while the ongoing cell divisions leave the cells as single cells. Late in the infection cycle, cells with low-density cell walls appear, and these cells can be detected on cytograms of light scatter versus, for instance, fluorescence of stained DNA. We describe a new method for detection of phage infection in
Lactococcus lactis
dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture. The method can be performed in real time and therefore increases the chance of successful intervention in the fermentation process.
Growth Characteristics of
Brevibacterium
,
Corynebacterium
,
Microbacterium
, and
Staphylococcus
spp. Isolated from Surface-Ripened Cheese
Authors: Mounier Jérôme , Rea Mary C. , O'Connor Paula M. , Fitzgerald Gerald F. , Cogan Timothy M.
Published: October 2007

The growth characteristics of five bacteria,
Brevibacterium aurantiacum
1-16-58,
Corynebacterium casei
DPC 5298
T
,
Corynebacterium variabile
DPC 5310,
Microbacterium gubbeenense
DPC 5286
T
, and
Staphylococcus saprophyticus
4E61, all of which were isolated from the surface of smear cheese, were studied in complex and chemically defined media. All of the coryneforms, except
M. gubbeenense
, grew in 12% salt, while
B. aurantiacum
and
S. saprophyticus
grew in 15% salt. All five bacteria assimilated lactate in a semisynthetic medium, and none of the coryneform bacteria assimilated lactose. Glucose assimilation was poor, except by
S. saprophyticus
and
C. casei
. Five to seven amino acids were assimilated by the coryneforms and 12 by
S. saprophyticus
. Glutamate, phenylalanine, and proline were utilized by all five bacteria, whereas utilization of serine, threonine, aspartate, histidine, alanine, arginine, leucine, isoleucine, and glycine depended on the organism. Growth of
C. casei
restarted after addition of glutamate, proline, serine, and lactate at the end of the exponential phase, indicating that these amino acids and lactate can be used as energy sources. Pantothenic acid was essential for the growth of
C. casei
and
M. gubbeenense
. Omission of biotin reduced the growth of
B. aurantiacum
,
C. casei
, and
M. gubbeenense
. All of the bacteria contained lactate dehydrogenase activity (with both pyruvate and lactate as substrates) and glutamate pyruvate transaminase activity but not urease activity.
Molecular Analysis of the Diversity of Sulfate-Reducing and Sulfur-Oxidizing Prokaryotes in the Environment, Using
aprA
as Functional Marker Gene
Authors: Meyer Birte , Kuever Jan
Published: October 2007

The dissimilatory adenosine-5′-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes,
aprBA
, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new
aprA
gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The
aprA
gene-based approach revealed putative sulfur-oxidizing
Alphaproteobacteria
of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the
Betaproteobacteria
and
Gammaproteobacteria
.
DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations
Authors: Plengvidhya Vethachai , Breidt Fredrick , Lu Zhongjing , Fleming Henry P.
Published: October 2007

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria,
Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus
, and
Lactobacillus brevis
, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include
Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis
, and
Weissella
sp. The newly identified species
Leuconostoc fallax
was also found. Unexpectedly, only two isolates of
P. pentosaceus
and 15 isolates of
L. brevis
were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.
N
-(3-Hydroxyhexanoyl)-
l
-Homoserine Lactone Is the Biologically Relevant Quormone That Regulates the
phz
Operon of
Pseudomonas chlororaphis
Strain 30-84
Authors: Khan Sharik R. , Herman Jake , Krank Jessica , Serkova Natalie J. , Churchill Mair E. A. , Suga Hiroaki , Farrand Stephen K.
Published: October 2007

Phenazine production by
Pseudomonas fluorescens
2-79 and
P. chlororaphis
isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from
P. fluorescens
2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these,
N
-(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of
P. chlororaphis
strains 30-84 and PCL1391 have been reported to produce and respond to
N
-(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by
P. chlororaphis
30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned
phzI
from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced
N
-(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated
N
-(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of
N
-(hexanoyl)-HSL. In titration assays, PhzR
30-84
responded to both
N
-(3-OH-hexanoyl)- and
N
-(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use
N
-(3-OH-hexanoyl)-HSL to activate PhzR.
Comparison of Cyanopeptolin Genes in
Planktothrix
,
Microcystis
, and
Anabaena
Strains: Evidence for Independent Evolution within Each Genus
Authors: Rounge Trine B. , Rohrlack Thomas , Tooming-Klunderud Ave , Kristensen Tom , Jakobsen Kjetill S.
Published: October 2007

The major cyclic peptide cyanopeptolin 1138, produced by
Planktothrix
strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb
oci
gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from
Anabaena
and
Microcystis
, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the
Planktothrix
and
Microcystis
cyanopeptolin domains than between these and the
Anabaena
domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.
Genome-Wide Screen ofSalmonellaGenes Expressed during Infection in Pigs, Using In Vivo Expression Technology
Authors: Huang Yanyan , Leming Christopher L. , Suyemoto Mitsu , Altier Craig
Published: October 2007

Pigs are a food-producing species that readily carrySalmonellabut, in the great majority of cases, do not show clinical signs of disease. Little is known about the functions required bySalmonellato be maintained in pigs. We have devised a recombinase-based promoter-trapping strategy to identify genes with elevated expression during pig infection withSalmonella entericaserovar Typhimurium. A total of 55 clones with in vivo-induced promoters were selected from a genomic library of ∼10,000 randomSalmonellaDNA fragments fused to the recombinasecre, and the cloned DNA fragments were analyzed by sequencing. Thirty-one genes encoding proteins involved in bacterial adhesion and colonization (includingbcfA, hscA, rffG, andyciR), virulence (metL), heat shock (hscA), and a sensor of a two-component regulator (hydH) were identified. Among the 55 clones, 19 were isolated from both the tonsils and the intestine, while 23 were identified only in the intestine and 13 only in tonsils. High temperature and increased osmolarity were identified as environmental signals that induced in vivo-expressed genes, suggesting possible signals for expression.