bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth Characteristics ofBartonella henselaein a Novel Liquid Medium: Primary Isolation, Growth-Phase-Dependent Phage Induction, and Metabolic Studies
Authors: Chenoweth M. R. , Somerville G. A. , Krause D. C. , O'Reilly K. L. , Gherardini F. C. 
Published: February 2004

ABSTRACTBartonella henselaeis a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolateB. henselaedirectly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 × 108CFU/ml) and permits the isolation ofB. henselaefrom the blood of infected cats. During the development of this medium, we observed thatB. henselaedid not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest,B. henselaedepleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phaseB. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed thatB. henselaehas a complete TCA cycle. Taken together, these data suggestB. henselaemay catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation ofB. henselaeand basic research into the pathogenesis of the bacterium.
Observing Growth and Division of Large Numbers of Individual Bacteria by Image Analysis
Authors: Elfwing A. , LeMarc Y. , Baranyi J. , Ballagi A. 
Published: February 2004

ABSTRACT
We describe a method that enabled us to observe large numbers of individual bacterial cells during a long period of cell growth and proliferation. We designed a flow chamber in which the cells attached to a transparent solid surface. The flow chamber was mounted on a microscope equipped with a digital camera. The shear force of the flow removed the daughter cells, making it possible to monitor the consecutive divisions of a single cell. In this way, kinetic parameters and their distributions, as well as some physiological characteristics of the bacteria, could be analyzed based on more than 1,000 single-cell observations. The method which we developed enabled us to study the history effect on the distribution of the lag times of single cells.
Genes Coding for Hepatotoxic Heptapeptides (Microcystins) in the Cyanobacterium
Anabaena
Strain 90
Authors: Rouhiainen Leo , Vakkilainen Tanja , Siemer Berit Lumbye , Buikema William , Haselkorn Robert , Sivonen Kaarina 
Published: February 2004

ABSTRACT

The cluster of microcystin synthetase genes from
Anabaena
strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (
mcyA
-
mcyB
-
mcyC
) is transcribed in the opposite direction from the second operon (
mcyG
-
mcyD
-
mcyJ
-
mcyE
-
mcyF
-
mcyI
) and the third operon (
mcyH
). The genes
mcyA
,
mcyB
, and
mcyC
encode nonribosomal peptide synthetases (NRPS), while
mcyD
codes for a polyketide synthase (PKS), and
mcyG
and
mcyE
are mixed NRPS-PKS genes. The genes
mcyJ
,
mcyF
, and
mcyI
are similar to genes coding for a methyltransferase, an aspartate racemase, and a
d
-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of
mcyB
coding for the adenylation domain was found to be 96% identical with the corresponding part of
mcyC
, suggesting a recent duplication of this fragment and a replacement in
mcyB
. In
Anabaena
strain 90, the order of the domains encoded by the genes in the two sets (from
mcyG
to
mcyI
and from
mcyA
to
mcyC
) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in
Anabaena
strain 90 differs from the arrangement found in two other cyanobacterial species,
Microcystis aeruginosa
and
Planktothrix agardhii
. The average sequence match between the microcystin synthetase genes of
Anabaena
strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.
Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom
Rhizosolenia setigera
Authors: Nagasaki Keizo , Tomaru Yuji , Katanozaka Noriaki , Shirai Yoko , Nishida Kensho , Itakura Shigeru , Yamaguchi Mineo 
Published: February 2004

ABSTRACT

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom
Rhizosolenia setigera
(
R. setigera
RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m
−2
s
−1
with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.
Variation in Sulfide Tolerance of Photosystem II in Phylogenetically Diverse Cyanobacteria from Sulfidic Habitats
Authors: Miller Scott R. , Bebout Brad M. 
Published: February 2004

ABSTRACT
Physiological and molecular phylogenetic approaches were used to investigate variation among 12 cyanobacterial strains in their tolerance of sulfide, an inhibitor of oxygenic photosynthesis. Cyanobacteria from sulfidic habitats were found to be phylogenetically diverse and exhibited an approximately 50-fold variation in photosystem II performance in the presence of sulfide. Whereas the degree of tolerance was positively correlated with sulfide levels in the environment, a strain's phenotype could not be predicted from the tolerance of its closest relatives. These observations suggest that sulfide tolerance is a dynamic trait primarily shaped by environmental variation. Despite differences in absolute tolerance, similarities among strains in the effects of sulfide on chlorophyll fluorescence induction indicated a common mode of toxicity. Based on similarities with treatments known to disrupt the oxygen-evolving complex, it was concluded that sulfide toxicity resulted from inhibition of the donor side of photosystem II.
Degradation Pathway and Generation of Monohydroxamic Acids from the Trihydroxamate Siderophore Deferrioxamine B
Authors: Pierwola Agnes , Krupinski Tomasz , Zalupski Peter , Chiarelli Michael , Castignetti Domenic 
Published: February 2004

ABSTRACT

Siderophores are avid ferric ion-chelating molecules that sequester the metal for microbes. Microbes elicit siderophores in numerous and different environments, but the means by which these molecules reenter the carbon and nitrogen cycles is poorly understood. The metabolism of the trihydroxamic acid siderophore deferrioxamine B by a
Mesorhizobium loti
isolated from soil was investigated. Specifically, the pathway by which the compound is cleaved into its constituent monohydroxamates was examined. High-performance liquid chromatography and mass-spectroscopy analyses demonstrated that
M. loti
enzyme preparations degraded deferrioxamine B, yielding a mass-to-charge (
m/z
) 361 dihydroxamic acid intermediate and an
m/z
219 monohydroxamate. The dihydroxamic acid was further degraded to yield a second molecule of the
m/z
219 monohydroxamate as well as an
m/z
161 monohydroxamate. These studies indicate that the dissimilation of deferrioxamine B by
M. loti
proceeds by a specific, achiral degradation and likely represents the reversal by which hydroxamate siderophores are thought to be synthesized.
A New Alkali-Thermostable Azoreductase from
Bacillus
sp. Strain SF
Authors: Maier Jürgen , Kandelbauer Andreas , Erlacher Angelika , Cavaco-Paulo Artur , Gübitz Georg M. 
Published: February 2004

ABSTRACT

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a
Bacillus
sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain,
Bacillus
sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80°C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a
Bacillus
sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(
R
)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO
2
substituents, especially in the
ortho
position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.
Lead Precipitation by
Vibrio harveyi
: Evidence for Novel Quorum-Sensing Interactions
Authors: Mire Chad E. , Tourjee Jeanette A. , O'Brien William F. , Ramanujachary Kandalam V. , Hecht Gregory B. 
Published: February 2004

ABSTRACT

Three pleiotropic, quorum sensing-defective
Vibrio harveyi
mutants were observed to precipitate soluble Pb
2+
as an insoluble compound. The compound was purified and subjected to X-ray diffraction and elemental analyses. These assays identified the precipitated compound as Pb
9
(PO
4
)
6
, an unusual and complex lead phosphate salt that is produced synthetically at temperatures of ca. 200°C. Regulation of the precipitation phenotype was also examined. Introduction of a
luxO
::
kan
allele into one of the mutants abolished lead precipitation, indicating that the well-characterized autoinducer 1 (AI1)-AI2 quorum-sensing system can block lead precipitation in dense cell populations. Interestingly, the
V. harveyi
D1 mutant, a strain defective for secretion of both AI1 and AI2, was shown to be an effective
trans
inhibitor of lead precipitation. This suggests that a previously undescribed
V. harveyi
autoinducer, referred to as AI3, can also negatively regulate lead precipitation. Experiments with heterologous bacterial populations demonstrated that many different species are capable of
trans
regulating the
V. harveyi
lead precipitation phenotype. Moreover, one of the
V. harveyi
mutants in this study exhibited little or no response to intercellular signals from other
V. harveyi
inocula but was quite responsive to some of the heterologous bacteria. Based on these observations, we propose that
V. harveyi
carries at least one quorum sensor that is specifically dedicated to receiving cross-species communication.
A Novel Saponin Hydrolase from
Neocosmospora vasinfecta
var.
vasinfecta
Authors: Watanabe Manabu , Sumida Naomi , Yanai Koji , Murakami Takeshi 
Published: February 2004

ABSTRACT

We isolated a soybean saponin hydrolase from
Neocosmospora vasinfecta
var.
vasinfecta
PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (
sdn1
) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The
sdn1
gene was successfully expressed in
Trichoderma viride
under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [α-
l
-rhamnopyranosyl (1→2)-β-
d
-galactopyranosyl (1→2)-
d
-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates.
Phenazines and Other Redox-Active Antibiotics Promote Microbial Mineral Reduction
Authors: Hernandez Maria E. , Kappler Andreas , Newman Dianne K. 
Published: February 2004

ABSTRACTNatural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction.Pseudomonas chlororaphisPCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures ofP. chlororaphisimplies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacteriumShewanella oneidensisMR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.
Shifts in the Membrane Fatty Acid Profile ofStreptococcus mutansEnhance Survival in Acidic Environments
Authors: Fozo Elizabeth M. , Quivey Robert G. 
Published: February 2004

ABSTRACTAcid adaptation ofStreptococcus mutansUA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of theS. mutansacid-adaptation arsenal.
Highly Stable
l
-Lysine 6-Dehydrogenase from the Thermophile
Geobacillus stearothermophilus
Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression
Authors: Heydari Mojgan , Ohshima Toshihisa , Nunoura-Kominato Naoki , Sakuraba Haruhiko 
Published: February 2004

ABSTRACT

l
-Lysine dehydrogenase, which catalyzes the oxidative deamination of
l
-lysine in the presence of NAD, was found in the thermophilic bacterium
Geobacillus stearothermophilus
UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ
1
-piperideine-6-carboxylate, indicating that the enzyme is
l
-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM
l
-lysine. The enzyme was relatively selective for
l
-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The

K
m

values for
l
-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in
Escherichia coli
, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of
G. stearothermophilus
. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.
Development of Additional Selectable Markers for the Halophilic ArchaeonHaloferax volcaniiBased on theleuBandtrpAGenes
Authors: Allers Thorsten , Ngo Hien-Ping , Mevarech Moshe , Lloyd Robert G. 
Published: February 2004

ABSTRACTSince most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry.Haloferax volcaniioffers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation ofH. volcanii leuBandtrpAgenes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuBand ΔtrpAmutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying theleuBortrpAgene under control of a constitutive promoter. The use of these selectable markers in deletion of thelhrgene ofH. volcaniiis described.
Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe
Authors: Eiden-Plach Antje , Zagorc Tatjana , Heintel Tanja , Carius Yvonne , Breinig Frank , Schmitt Manfred J. 
Published: February 2004

ABSTRACTBesides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1andTPI1) or from inducible and/or repressible (GAL1,AOX1, andNMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.
Selection of Unusual Actinomycetal Primary σ
70
Factors by Plant-Colonizing
Frankia
Strains
Authors: Lavire Céline , Blaha Didier , Cournoyer Benoit 
Published: February 2004

ABSTRACT

Functional adaptations of σ
70
transcriptional factors led to the emergence of several paralogous lineages, each one being specialized for gene transcription under particular growth conditions. Screening of a
Frankia
strain EaI-12 gene library by σ
70
DNA probing allowed the detection and characterization of a novel actinomycetal primary (housekeeping) σ
70
factor. Phylogenetic analysis positioned this factor in the RpoD cluster of proteobacterial and low-G+C-content gram-positive factors, a cluster previously free of any actinobacterial sequences. σ
70
DNA probing of
Frankia
total DNA blots and PCR screening detected one or two
rpoD
-like DNA regions per species.
rpoD
matched the conserved region in all of the species tested. The other region was found to contain
sigA
, an alternative primary factor.
sigA
appeared to be strictly distributed among
Frankia
species infecting plants by the root hair infection process. Both genes were transcribed by
Frankia
strain ACN14a grown in liquid cultures. The molecular phylogeny of the σ
70
family determined with
Frankia
sequences showed that the alternative actinomycetal factors and the essential ones belonged to the same radiation. At least seven distinct paralogous lineages were observed among this radiation, and gene transfers were detected in the HrdB actinomycetal lineage.