bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective Efficacy of a Sulfated Sialyl Lipid (NMSO3) against Human Rotavirus-Induced Diarrhea in a Mouse Model
Authors: Takahashi Kazuo , Ohashi Kazutaka , Abe Yurika , Mori Shuichi , Taniguchi Koki , Ebina Takusaburo , Nakagomi Osamu , Terada Masaki , Shigeta Shiro
Published: July 2002

Antiviral activity of sulfated sialyl lipid (NMSO3) against human rotavirus (RV) was examined in vitro and in vivo. NMSO3 inhibited the replication of four major serotypes (G1 to G4) of human rotavirus with a low 50% effective concentration of 1 to 5 μg/ml and 50% cytotoxic concentration of 153 μg/ml when determined by plaque assays with MA104 cells. Exposure of NMSO3 to HCl (pH 2.0) for 30 min exhibited no loss of anti-RV activity. Time-of-addition experiments revealed that NMSO3 inhibited the adsorption of four serotypes of RV to MA104 cells. Furthermore, an assay of virus binding with radiolabeled RVs revealed that NMSO3 inhibited the binding of virus to MA104 cells, suggesting that NMSO3 may bind to VP4 and/or VP7. Prophylactic oral administration of NMSO3 (10 μg three times per day, 4 days) to five suckling mice starting 30 min before inoculation of MO strain (3 × 106PFU/mouse) prevented the development of diarrhea. Four of five mice showed no stool or brown formed stool, and only one mouse showed brown soft stool, while water treatment caused watery diarrhea in all five mice. The mean titer of antibody to RV in mice which received NMSO3 at 10 μg three times per day for 4 days was significantly lower than that of untreated, infected mice. NMSO3 is a promising candidate for the prophylactic treatment of human RVs.
Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of
Acanthamoeba
spp
Authors: Walochnik Julia , Duchêne Michael , Seifert Karin , Obwaller Andreas , Hottkowitz Thomas , Wiedermann Gerhard , Eibl Hansjörg , Aspöck Horst
Published: July 2002

Free-living amoebae of the genus
Acanthamoeba
are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against
Acanthamoeba
spp. The in vitro activities of eight different APCs against three
Acanthamoeba
strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of
Acanthamoeba
keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.
Biosynthetic Gene Cluster of Simocyclinone, a Natural Multihybrid Antibiotic
Authors: Trefzer A. , Pelzer S. , Schimana J. , Stockert S. , Bihlmaier C. , Fiedler H.-P. , Welzel K. , Vente A. , Bechthold A.
Published: July 2002

The entire simocyclinone biosynthetic cluster (
sim
gene cluster) from the producer
Streptomyces antibioticus
Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the
sim
gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the
sim
gene cluster had been cloned.
Bloodstream Infections by Extended-Spectrum β-Lactamase-Producing
Escherichia coli
and
Klebsiella pneumoniae
in Children: Epidemiology and Clinical Outcome
Authors: Kim Yun-Kyung , Pai Hyunjoo , Lee Hoan-Jong , Park Su-Eun , Choi Eun-Hwa , Kim Jungmin , Kim Je-Hak , Kim Eui-Chong
Published: July 2002

To determine the epidemiologic features and clinical outcomes of bloodstream infections caused by extended-spectrum β-lactamase (ESBL)-producing
Escherichia coli
and
Klebsiella pneumoniae
isolates, cases of bacteremia caused by these organisms in children were analyzed retrospectively. Among the 157 blood isolates recovered from 1993 to 1998 at the Seoul National University Children's Hospital, the prevalence of ESBL production was 17.9% among the
E. coli
isolates and 52.9% among the
K. pneumoniae
isolates. The commonest ESBLs were SHV-2a and TEM-52. A novel ESBL, TEM-88, was identified. Pulsed-field gel electrophoresis analysis of the ESBL-producing organisms showed extensive diversity in clonality. The medical records of 142 episodes were reviewed. The risk factors for bloodstream infection with ESBL-producing organisms were prior hospitalization, prior use of oxyimino-cephalosporins, and admission to an intensive care unit within the previous month. There was no difference in clinical severity between patients infected with ESBL-producing strains (the ESBL group) and those infected with ESBL-nonproducing strains (the non-ESBL group) at the time of presentation. However, the overall fatality rate for the ESBL group was significantly higher than that for the non-ESBL group: 12 of 45 (26.7%) versus 5 of 87 (5.7%) (
P
= 0.001). In a subset analysis of patients treated with extended-spectrum cephalosporins with or without an aminoglycoside, favorable response rates were significantly higher in the non-ESBL group at the 3rd day (6 of 17 versus 33 of 51;
P
= 0.035), the 5th day (6 of 17 versus 36 of 50;
P
< 0.05), and the end of therapy (9 of 17 versus 47 of 50;
P
< 0.001). In conclusion, the ESBL production of the infecting organisms has a significant impact on the clinical course and survival of pediatric patients with bacteremia caused by
E. coli
and
K. pneumoniae
.
Tenofovir Diphosphate Is a Poor Substrate and a Weak Inhibitor of Rat DNA Polymerases α, δ, and ε*
Authors: Birkuš Gabriel , Hájek Miroslav , Kramata Pavel , Votruba Ivan , Holý Antonín , Otová Berta
Published: July 2002

Tenofovir diphosphate (PMPApp) is a weak inhibitor of DNA polymerases (pol) α, δ, and ε*, with values for the

K
i

for PMPApp (
PMPApp

K
i

) relative to the

K
m

for dATP (
dATP

K
m

) of 10.2, 10.2, and 15.2, respectively. Its incorporation into DNA was about 1,000-fold less efficient than that of dATP, with
PMPApp

K
m

values 350-, 2,155-, and 187-fold higher than
dATP

K
m

values for pol α, δ, and ε*, respectively.
Microbial Degradation of the Multiply Branched Alkane 2,6,10,15,19,23-Hexamethyltetracosane (Squalane) by
Mycobacterium fortuitum
and
Mycobacterium ratisbonense
Authors: Berekaa Mahmoud M. , Steinbüchel Alexander
Published: July 2002

Among several bacterial species belonging to the general
Gordonia
,
Mycobacterium
,
Micromonospora
,
Pseudomonas
, and
Rhodococcus
, only two mycobacterial isolates,
Mycobacterium fortuitum
strain NF4 and the new isolate
Mycobacterium ratisbonense
strain SD4, which was isolated from a sewage treatment plant, were capable of utilizing the multiply branched hydrocarbon squalane (2,6,10,15,19,23-hexamethyltetracosane) and its analogous unsaturated hydrocarbon squalene as the sole carbon source for growth. Detailed degradation studies and high-pressure liquid chromatography analysis showed a clear decrease of the concentrations of squalane and squalene during biomass increase. These results were supported by resting-cell experiments using strain SD4 and squalane or squalene as the substrate. The degradation of acyclic isoprenoids and alkanes as well as of acids derived from these compounds was also investigated. Inhibition of squalane and squalene degradation by acrylic acid indicated the possible involvement of β-oxidation in the degradation route. To our knowledge, this is the first report demonstrating the biodegradation of squalane by using defined axenic cultures.
adhA
in
Aspergillus parasiticus
Is Involved in Conversion of 5′-Hydroxyaverantin to Averufin
Authors: Chang Perng-Kuang , Yu Jiujiang , Ehrlich Kenneth C. , Boue Stephen M. , Montalbano Beverly G. , Bhatnagar Deepak , Cleveland Thomas E.
Published: July 2002

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene,
adhA
, from the aflatoxin gene cluster of
Aspergillus parasiticus
SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD
+
-NADP
+
, and the motif YXXXK, which is found at the active site.
A. parasiticus
SU-1, which produces aflatoxins, has two copies of
adhA
(
adhA1
), whereas
A. parasiticus
SRRC 2043, a strain that accumulates
O
-methylsterigmatocystin (OMST), has only one copy. Disruption of
adhA
in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those
adhA
disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.
Thermostable NADP
+
-Dependent Medium-Chain Alcohol Dehydrogenase from
Acinetobacter
sp. Strain M-1: Purification and Characterization and Gene Expression in
Escherichia coli
Authors: Tani Akio , Sakai Yasuyoshi , Ishige Takeru , Kato Nobuo
Published: July 2002

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by
n
-hexadecane, from
Acinetobacter
sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward
n
-heptanal and could use
n
-alkylaldehydes ranging from C
2
to C
14
and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The
alrA
gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the
alrA
gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in
Escherichia coli
, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.
Spatial Changes in the Bacterial Community Structure along a Vertical Oxygen Gradient in Flooded Paddy Soil Cores
Authors: Lüdemann Heiner , Arth Inko , Liesack Werner
Published: July 2002

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 μM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-μm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the α and β subclasses of
Proteobacteria
as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.
Immunolocalization of Dinitrogenase Reductase Produced by
Klebsiella pneumoniae
in Association with
Zea mays
L
Authors: Chelius Marisa K. , Triplett Eric W.
Published: July 2002

The endophytic lifestyle of
Klebsiella pneumoniae
is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by
K. pneumoniae
strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for
K. pneumoniae
and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.
Characterization and Determination of Origin of Lactic Acid Bacteria from a Sorghum-Based Fermented Weaning Food by Analysis of Soluble Proteins and Amplified Fragment Length Polymorphism Fingerprinting
Authors: Kunene Nokuthula F. , Geornaras Ifigenia , von Holy Alexander , Hastings John W.
Published: July 2002

The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains were
Lactobacillus plantarum
strains, 47 were
Leuconostoc mesenteroides
strains, 25 were
Lactobacillus sake-Lactobacillus curvatus
strains, 17 were
Pediococcus pentosaceus
strains, 13 were
Pediococcus acidilactici
strains, and 7 were
Lactococcus lactis
strains.
L. plantarum
and
L. mesenteroides
strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.
Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions
Authors: Barth S. , Huhn M. , Matthey B. , Klimka A. , Galinski E. A. , Engert A.
Published: July 2002

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of
Escherichia coli
. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of
E. coli
under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.
Long-Chain Aldehyde Dehydrogenase That Participates in
n
-Alkane Utilization and Wax Ester Synthesis in
Acinetobacter
sp. Strain M-1
Authors: Ishige Takeru , Tani Akio , Sakai Yasuyoshi , Kato Nobuo
Published: July 2002

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of
Acinetobacter
sp. strain M-1 cells grown on
n
-hexadecane as a sole carbon source. The gene (
ald1
) was cloned from the chromosomal DNA of the bacterium. The open reading frame of
ald1
was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The
ald1
gene was stably expressed in
Escherichia coli
, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward
n
-alkanals (C
4
to C
14
), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The
ald1
gene was disrupted by homologous recombination on the
Acinetobacter
genome. Although the
ald1
disruptant (
ald1Δ
) strain still had the ability to grow on
n
-hexadecane to some extent, its aldehyde dehydrogenase activity toward
n
-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the
ald1
Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on
n
-alkane and in wax ester formation in
Acinetobacter
sp. strain M-1.
Cold Shock Proteins ofLactococcus lactisMG1363 Are Involved in Cryoprotection and in the Production of Cold-Induced Proteins
Authors: Wouters Jeroen A. , Frenkiel Hélène , de Vos Willem M. , Kuipers Oscar P. , Abee Tjakko
Published: July 2002

Members of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacteriumLactococcus lactisMG1363. By using double-crossover recombination, twoL. lactisstrains were generated in which genes encoding CSPs are disrupted:L. lactisNZ9000ΔAB lacks the tandemly orientatedcspAandcspBgenes, and NZ9000ΔABE lackscspA, cspB, andcspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain,L. lactisNZ9000. Two-dimensional gel electrophoresis showed that upon disruption of thecspABgenes, the production of remaining CspE at low temperature increased, and upon disruption ofcspA, cspB, andcspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in thecsp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756–3763, 2000). that the CSPs ofL. lactismight be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10°C was significantly reduced in strain NZ9000ΔABE but not in strain NZ9000ΔAB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.
Purification and Properties of a Glucuronan Lyase from
Sinorhizobium meliloti
M5N1CS (NCIMB 40472)
Authors: Da Costa Alexandre , Michaud Philippe , Petit Emmanuel , Heyraud Alain , Colin-Morel Philippe , Courtois Bernard , Courtois Josiane
Published: July 2002

A glucuronan lyase extracted from
Sinorhizobium meliloti
strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn
2+
, Cu
2+
, and Hg
2+
(1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from
S. meliloti
strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.
In Planta Regulation of Extension of an Endophytic Fungus and Maintenance of High Metabolic Rates in Its Mycelium in the Absence of Apical Extension
Authors: Tan Yong Y. , Spiering Martin J. , Scott Vicki , Lane Geoffrey A. , Christensen Michael J. , Schmid Jan
Published: July 2002

The fungus
Neotyphodium lolii
is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass
Lolium perenne,
producing secondary metabolites which enhance the fitness of the association over that of uninfected
L. perenne
. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.
Novel Zn
2+
-Chelating Peptides Selected from a Fimbria-Displayed Random Peptide Library
Authors: Kjærgaard Kristian , Schembri Mark A. , Klemm Per
Published: July 2002

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of
Escherichia coli
which mediate
d
-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the
E. coli
cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of ∼40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn
2+
. By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn
2+
were enriched. None of the isolated sequences showed similarity to known Zn
2+
-binding proteins, indicating that completely novel Zn
2+
-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn
2+
-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.
Influence of Tomato Genotype on Growth of Inoculated and Indigenous Bacteria in the Spermosphere
Authors: Simon Holly M. , Smith Kevin P. , Dodsworth Jeremy A. , Guenthner Bobbi , Handelsman Jo , Goodman Robert M.
Published: July 2002

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent,
Bacillus cereus
UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786–4790, 1999). Here we report results of studies examining the host effect on the support of growth of
Bacillus
and
Pseudomonas
strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of
B. cereus
UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24
B. cereus
strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of
B. cereus
UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains
Pseudomonas fluorescens
2-79 or
Pseudomonas aureofaciens
AB254. Thus, while the host effect on growth extended to all strains of
B. cereus
examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of
B. cereus
UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous
Bacillus
and fluorescent
Pseudomonas
bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous
Bacillus
soil bacteria, while the line that supported growth of inoculated
B. cereus
UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of
B. cereus
UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains—both
Bacillus
and
Pseudomonas
spp.—did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.
Analysis of the Genetic Switch and Replication Region of a P335-Type Bacteriophage with an Obligate Lytic Lifestyle on
Lactococcus lactis
Authors: Madsen Søren M. , Mills David , Djordjevic Gordana , Israelsen Hans , Klaenhammer Todd R.
Published: July 2002

The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage φ31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that φ31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a
lacLM
reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate
Lactococcus lactis
phages. The
cro
gene homologue regulates its own production and is an efficient repressor of
c
I gene expression. No detectable
c
I gene expression could be measured in the presence of
cro. c
I gene expression in the absence of
cro
exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the
cro
gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several
Streptococcus thermophilus
phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of φ31 10
6
-fold when present at high copy number in
trans
provide evidence for identification of the phage origin of replication. Phage φ31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of
Lactococcus
,
Lactobacillus
, and
Streptococcus
were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.
Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains
Authors: Hickey Rita M. , Twomey Denis P. , Ross R. Paul , Hill Colin
Published: July 2002

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (
oriT
). This
oriT
-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10
−4
transconjugants/donor cell from an MG1363 (
recA
mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the
oriT
-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that
oriT
of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.
Distribution of Archaea in a Black Smoker Chimney Structure
Authors: Takai Ken , Komatsu Tetsushi , Inagaki Fumio , Horikoshi Koki
Published: July 2002

Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were evaluated through the combined use of culture-independent molecular analyses and enrichment culture methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua New Guinea, and subsamples were obtained from vertical and horizontal sections. The elemental composition of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of elemental oxygen in exterior materials represented the presence of oxidized materials on the outer surface of the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal ribotype structure occurred in the chimney structure. Through sequencing of ribosomal DNA (rDNA) clones from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the distribution of such extremophiles in different microhabitats of the chimney varied. The results of the culture-dependent analysis supported in part the view that changes in archaeal community structures in these microhabitats are associated with the geochemical and physical dynamics in the black smoker chimney.
Occurrence ofCryptosporidiumandGiardiain Wild Ducks along the Rio Grande River Valley in Southern New Mexico
Authors: Kuhn Ryan C. , Rock Channah M. , Oshima Kevin H.
Published: July 2002

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001.Giardiacysts andCryptosporidiumoocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence ofGiardia lambliaandCrytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers ofCryptosporidium, and theCryptosporidiumoocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ± standard deviation, 47.53 ± 270.3 oocysts per g); also, 28% of the ducks were positive forGiardia, and theGiardiacyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ± standard deviation, 436 ± 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive forCryptosporidiumsp. However,C. parvumandG. lambliawere not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive forCryptosporidiumandGiardiaas determined by FA staining, butC. parvumandG. lambliawere not detected.
Characterization of an Autotrophic Sulfide-Oxidizing Marine
Arcobacter
sp. That Produces Filamentous Sulfur
Authors: Wirsen C. O. , Sievert S. M. , Cavanaugh C. M. , Molyneaux S. J. , Ahmad A. , Taylor L. T. , DeLong E. F. , Taylor C. D.
Published: July 2002

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus
Arcobacter
in the epsilon subdivision of the
Proteobacteria
. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with
Arcobacter
-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “
Candidatus
Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO
2
fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as
Thiomicrospira
spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO
2
fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “
Candidatus
Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.
Staphylococcus aureusGrowth Boundaries: Moving towards Mechanistic Predictive Models Based on Solute-Specific Effects
Authors: Stewart Cynthia M. , Cole Martin B. , Legan J. David , Slade Louise , Vandeven Mark H. , Schaffner Donald W.
Published: July 2002

The formulation of shelf-stable intermediate-moisture products is a critical food safety issue. Therefore, knowing the precise boundary for the growth-no-growth interface ofStaphylococcus aureusis necessary for food safety risk assessment. This study was designed to examine the effects of various humectants and to produce growth boundary models as tools for risk assessment. The molecular mobility and the effects of various physical properties of humectants, such as their glass transition temperatures, their membrane permeability, and their ionic and nonionic properties, onS. aureusgrowth were investigated. The effects of relative humidity (RH; 84 to 95%, adjusted by sucrose plus fructose, glycerol, or NaCl), initial pH (4.5 to 7.0, adjusted by HCl), and potassium sorbate concentration (0 or 1,000 ppm) on the growth ofS. aureuswere determined. Growth was monitored by turbidity over a 24-week period. Toxin production was determined by enterotoxin assay. The 1,792 data points generated were analyzed by LIFEREG procedures (SAS Institute, Inc., Cary, N.C.), which showed that all parameters studied significantly affected the growth responses ofS. aureus. Differences were observed in the growth-no-growth boundary when different humectants were used to achieve the desired RH values in both the absence and the presence of potassium sorbate. Sucrose plus fructose was most inhibitory at neutral pH values, while NaCl was most inhibitory at low pH values. The addition of potassium sorbate greatly increased the no-growth regions, particularly when pH was <6.0. Published kinetic growth and survival models were compared with boundary models developed in this work. The effects of solutes and differences in modeling approaches are discussed.
Fasciclin Domain Proteins Are Present in
Nostoc
Symbionts of Lichens
Authors: Paulsrud Per , Lindblad Peter
Published: July 2002

Differences in the soluble protein fraction between the freshly isolated cyanobiont of lichen
Peltigera membranacea
, the corresponding free-living strain, and
Nostoc punctiforme
were analyzed. One protein, which was among the most prominent proteins of the freshly isolated cyanobiont, was expressed at a lower level in the corresponding free-living strain and was not detected at all on the two-dimensional gels of
N. punctiforme
. This protein was partially sequenced, and the corresponding open reading frame (ORF) in the
N. punctiforme
genome was identified. This ORF contains a fasciclin domain typical of a class of surface-associated proteins involved in cell adhesion. Similar fasciclin motif-containing genes have previously been shown to be symbiotically induced in other symbiotic systems.
Tri13
and
Tri7
Determine Deoxynivalenol- and Nivalenol-Producing Chemotypes of
Gibberella zeae
Authors: Lee Theresa , Han You-Kyoung , Kim Kook-Hyung , Yun Sung-Hwan , Lee Yin-Won
Published: July 2002

Gibberella zeae
, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a
Tri13
homolog from each chemotype. The
Tri13
from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of
Fusarium sporotrichioides
, whereas the
Tri13
from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the
Tri13
and
Tri7
genes in trichothecene production by
G. zeae
, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of
Tri13
from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1
Tri13
gene alone or in combination with the 88-1
Tri7
gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that
Tri13
and
Tri7
are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in
G. zeae
. These functional analyses of the
Tri13
and
Tri7
genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in
G. zeae
.
Influence of Different Cultivars on Populations of Ammonia-Oxidizing Bacteria in the Root Environment of Rice
Authors: Briones Aurelio M. , Okabe Satoshi , Umemiya Yoshiaki , Ramsing Niels-Birger , Reichardt Wolfgang , Okuyama Hidetoshi
Published: July 2002

Comparisons of the activities and diversities of ammonia-oxidizing bacteria (AOB) in the root environment of different cultivars of rice (
Oryza sativa
L.) indicated marked differences despite identical environmental conditions during growth. Gross nitrification rates obtained by the
15
N dilution technique were significantly higher in a modern variety, IR63087-1-17, than in two traditional varieties. Phylogenetic analysis based on the ammonium monooxygenase gene (
amoA
) identified strains related to
Nitrosospira multiformis
and
Nitrosomonas europaea
as the predominant AOB in our experimental rice system. A method was developed to determine the abundance of AOB on root biofilm samples using fluorescently tagged oligonucleotide probes targeting 16S rRNA. The levels of abundance detected suggested an enrichment of AOB on rice roots. We identified 40 to 69% of AOB on roots of IR63087-1-17 as
Nitrosomonas
spp., while this subpopulation constituted 7 to 23% of AOB on roots of the other cultivars. These results were generally supported by denaturing gradient gel electrophoresis of the
amoA
gene and analysis of libraries of cloned
amoA
. In hydroponic culture, oxygen concentration profiles around secondary roots differed significantly among the tested rice varieties, of which IR63087-1-17 showed maximum leakage of oxygen. The results suggest that varietal differences in the composition and activity of root-associated AOB populations may result from microscale differences in O
2
availability.
Enterotoxin Production in Natural Isolates of
Bacillaceae
outside the
Bacillus cereus
Group
Authors: Phelps Rebecca J. , McKillip John L.
Published: July 2002

Thirty-nine
Bacillus
strains obtained from a variety of environmental and food sources were screened by PCR for the presence of five gene targets (
hblC
,
hblD
,
hblA
,
nheA
, and
nheB
) in two enterotoxin operons (HBL and NHE) traditionally harbored by
Bacillus cereus
. Seven isolates exhibited a positive signal for at least three of the five possible targets, including
Bacillus amyloliquefaciens
,
B. cereus
,
Bacillus circulans
,
Bacillus lentimorbis
,
Bacillus pasteurii
, and
Bacillus thuringiensis
subsp.
kurstaki
. PCR amplicons were confirmed by restriction enzyme digest patterns compared to a positive control strain. Enterotoxin gene expression of each strain grown in a model food system (skim milk) was monitored by gene-specific reverse transcription-PCR and confirmed with the Oxoid RPLA and Tecra BDE commercial kits. Lecithinase production was noted on egg yolk-polymyxin B agar for all strains except
B. lentimorbis
, whereas discontinuous beta hemolysis was exhibited by all seven isolates grown on 5% sheep blood agar plates. The results of this study confirm the presence of enterotoxin genes in natural isolates of
Bacillus
spp. outside the
B. cereus
group and the ability of these strains to produce toxins in a model food system under aerated conditions at 32°C.
Comparison of Five Commercial Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to
Bordetella pertussis
Authors: Kösters Katrin , Riffelmann Marion , Dohrn Brigitte , von König Carl Heinz Wirsing
Published: July 2002

Measuring antibodies to
Bordetella pertussis
antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.
Peptidoglycan and Lipoteichoic Acid Modify Monocyte Phenotype in Human Whole Blood
Authors: Jørgensen Pål F. , Wang Jacob E. , Almlöf Mia , Thiemermann Christoph , Foster Simon J. , Solberg Rigmor , Aasen Ansgar O.
Published: July 2002

We examined the influence of the gram-positive cell wall products peptidoglycan (PepG) and lipoteichoic acid (LTA), compared to lipopolysaccharide (LPS), on the monocyte expression of receptors involved in antigen presentation (HLA-DR, B7.1, and B7.2), cell adhesion (intercellular adhesion molecule-1 [ICAM-1] and lymphocyte function associated antigen-3 [LFA-3]), phagocytosis (FcγRI), and cell activation (CD14). We also evaluated possible influences of the immunosuppressive drugs cyclosporine A, tacrolimus, and sirolimus on the expression of these receptors. Pretreatment of whole blood for 4 h with the immunosuppressive drugs did not influence the expression of the surface receptors in normal or stimulated blood. Stimulation with both PepG and LTA caused significant up-regulation of the surface expression of ICAM-1 and HLA-DR on whole blood monocytes, similar to that obtained with LPS, whereas B7.1, B7.2, LFA-3, and FcγRI were not modulated. PepG and LTA also caused increased expression of CD14, whereas LPS down-regulated this molecule. In contrast, we did not detect any significant influence of any of the bacterial products on the plasma concentration of soluble CD14. We hypothesized that the increased expression of surface CD14 in blood stimulated with PepG would prime for cellular activation by LPS. Indeed, we show that PepG and the partial PepG structure muramyl dipeptide acted in synergy with LPS to cause the release of tumor necrosis factor-α. The results suggest that PepG and LPS provoke partly different responses on monocyte phenotype and that CD14 may play different roles in the innate response to gram-positive and gram-negative bacteria.