bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Killing of
Bacillus subtilis
on the Hair Roots of Transgenic T4 Lysozyme-Producing Potatoes
Authors: Ahrenholtz Ingrid , Harms Klaus , de Vries Johann , Wackernagel Wilfried
Published: July 2002

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium
Erwinia carotovora
subsp.
carotovora
have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of
Bacillus subtilis
with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed
B. subtilis
cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.
Cospeciation of Psyllids and Their Primary Prokaryotic Endosymbionts
Authors: Thao MyLo L. , Moran Nancy A. , Abbot Patrick , Brennan Eric B. , Burckhardt Daniel H. , Baumann Paul
Published: July 2002

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene
wingless
were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host
wingless
gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3′ end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain
Bacteria
in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation
Candidatus
Carsonella (gen. nov.) with a single species,
Candidatus
Carsonella ruddii (sp. nov.).
A Genomic Sample Sequence of the Entomopathogenic Bacterium
Photorhabdus luminescens
W14: Potential Implications for Virulence
Authors: Ffrench-Constant Richard H. , Waterfield Nicholas , Burland Valerie , Perna Nicole T. , Daborn Phillip J. , Bowen David , Blattner Frederick R.
Published: July 2002

Photorhabdus luminescens
is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this “symbiosis of pathogens” remain largely obscure. In order to identify genes encoding potential virulence factors, we performed ∼2,000 random sequencing reads from a
P. luminescens
W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the
Escherichia coli
K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen
P. luminescens
shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.
Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic
Arabidopsis
Plants
Authors: Yuan Qiaoping , Hu Wenqi , Pestka James J. , He Sheng Yang , Hart L. Patrick
Published: July 2002

The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using
Arabidopsis
as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S
2
. After transformation, constructs of antizearalenone scFv were introduced into immature
Arabidopsis
seeds via
Agrobacterium tumefaciens
mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv “plantibody” from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.
Production of Poly(3-Hydroxybutyrate) by Fed-Batch Culture of Recombinant
Escherichia coli
with a Highly Concentrated Whey Solution
Authors: Ahn Woo Suk , Park Si Jae , Lee Sang Yup
Published: July 2002

Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant
Escherichia coli
strain CGSC 4401 harboring the
Alcaligenes latus
polyhydroxyalkanoate (PHA) biosynthesis genes were developed. The pH-stat fed-batch cultures of
E. coli
CGSC 4401 harboring pJC4, a stable plasmid containing the
A. latus
PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter. Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.
Identification of a Novel Metabolite in the Degradation of Pyrene by
Mycobacterium
sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons
Authors: Vila Joaquim , López Zaira , Sabaté Jordi , Minguillón Cristina , Solanas Anna M. , Grifoll Magdalena
Published: July 2002

Mycobacterium
sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give
trans-
or
cis-
4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter,
ortho
cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and
ortho
cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of
ortho
cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.
Characteristics of a
Streptomyces coelicolor
A3(2) Extracellular Protein Targeting Chitin and Chitosan
Authors: Saito Akihiro , Miyashita Kiyotaka , Biuković Goran , Schrempf Hildgund
Published: July 2002

Upstream of the
Streptomyces coelicolor
A3(2) chitinase G gene, a small gene (named
chb3
) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from
Streptomyces olivaceoviridis
. The
chb3
gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free
Streptomyces lividans
TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the
S. lividans
TK21 transformant during growth in the presence of glucose,
N
-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets α-chitin, β-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.
Quorum-Sensing Genes in
Pseudomonas aeruginosa
Biofilms: Their Role and Expression Patterns
Authors: De Kievit Teresa R. , Gillis Richard , Marx Steve , Brown Chris , Iglewski Barbara H.
Published: July 2002

Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). In
Pseudomonas aeruginosa
, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the
las
and
rhl
QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of
lasI
and
rhlI
to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development,
lasI
expression was found to progressively decrease over time. Conversely,
rhlI
expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that
lasI
and
rhlI
were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.
Genotypic Analysis of
Escherichia coli
Strains from Poultry Carcasses and Their Susceptibilities to Antimicrobial Agents
Authors: Geornaras Ifigenia , Hastings John W. , von Holy Alexander
Published: July 2002

Plasmid profiling and amplified fragment length polymorphism (AFLP) analysis were used to genotype 50
Escherichia coli
strains from poultry carcasses. Thirty different plasmid profiles were evident, and clustering of the AFLP data showed that they were a distinctly heterogeneous group of strains. Susceptibility testing against five antimicrobial agents used in the South African poultry industry showed all strains to be susceptible to danofloxacin and colistin, while the majority (96%) were resistant to two tetracyclines.
Spontaneous Formation of a Mannitol-Producing Variant of
Leuconostoc pseudomesenteroides
Grown in the Presence of Fructose
Authors: Grobben Gert J. , Peters Sjors W. P. G. , Wisselink H. Wouter , Weusthuis Ruud A. , Hoefnagel Marcel H. N. , Hugenholtz Jeroen , Eggink Gerrit
Published: July 2002

We report the spontaneous formation of a stable mannitol-producing variant of
Leuconostoc pseudomesenteroides
. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.
Chlorine Dioxide Inactivation of
Cryptosporidium parvum
Oocysts and Bacterial Spore Indicators
Authors: Chauret Christian P. , Radziminski Chris Z. , Lepuil Michael , Creason Robin , Andrews Robert C.
Published: July 2002

Cryptosporidium parvum
, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against
Cryptosporidium
oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (
P
< 0.05). Products of disinfectant concentration and contact time (
Ct
values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log
10
and 2.0 log
10
units (99% inactivation) of
C. parvum
as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide.
Ct
values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log
10
units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including
Bacillus subtilis
(aerobic) spores and
Clostridium sporogenes
(anaerobic) spores, and
C. parvum
oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than
C. parvum
oocysts and therefore could not be used as direct indicators of
C. parvum
inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of
C. parvum
, since these factors might affect oocyst disinfection sensitivity.
Adhesion of Bifidobacteria to Granular Starch and Its Implications in Probiotic Technologies
Authors: Crittenden R. , Laitila A. , Forssell P. , Mättö J. , Saarela M. , Mattila-Sandholm T. , Myllärinen P.
Published: July 2002

Adhesion of 19
Bifidobacterium
strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (
Bifidobacterium adolescentis
VTT E-001561 and
Bifidobacterium pseudolongum
ATCC 25526), adhesion appeared to be specific for α-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of ≤3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.
Methane Oxidation and the Competition for Oxygen in the Rice Rhizosphere
Authors: van Bodegom Peter , Stams Fons , Mollema Liesbeth , Boeke Sara , Leffelaar Peter
Published: July 2002

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O
2
with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a
Methylocystis
sp.) and heterotrophs (a
Pseudomonas
sp. and a
Rhodococcus
sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently <5 μM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between
Pseudomonas
spp. and
Methylocystis
spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.
Effectiveness of Germicidal UV Radiation for Reducing Fungal Contamination within Air-Handling Units
Authors: Levetin Estelle , Shaughnessy Richard , Rogers Christine A. , Scheir Robert
Published: July 2002

Levels of fungi growing on insulation within air-handling units (AHUs) in an office building and levels of airborne fungi within AHUs were measured before the use of germicidal UV light and again after 4 months of operation. The fungal levels following UV operation were significantly lower than the levels in control AHUs.
Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of
Clostridium cellulolyticum
on a Chemically Defined Medium
Authors: Desvaux Mickaël , Guedon Emmanuel , Petitdemange Henri
Published: July 2002

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of
Clostridium cellulolyticum
with increasing amounts of carbon substrate. At a dilution rate (
D
) of 0.048 h
−1
, biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose liter
−1
the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose liter
−1
, respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of
C. cellulolyticum
metabolism in cellulose saturation was performed by varying
D
with 18.1 g of cellulose liter
−1
. Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119–130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD
+
, and
q
NADH produced
/
q
NADH used
ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as
D
rose, and (iii) the maximum growth yield ( Y

X
/
S

max

) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield ( Y

ATP

max

) (19.4 g of biomass per mol of ATP) were lowered.
C. cellulolyticum
was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327–335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells
−1
h
−1
could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells
−1
h
−1
; (ii) while the NADH/NAD
+
ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.
Antimicrobial Properties of Pyridine-2,6-Dithiocarboxylic Acid, a Metal Chelator Produced by
Pseudomonas
spp
Authors: Sebat J. L. , Paszczynski A. J. , Cortese M. S. , Crawford R. L.
Published: July 2002

Pyridine-2,6-dithiocarboxylic acid (pdtc) is a metal chelator produced by
Pseudomonas
spp. It has been shown to be involved in the biodegradation of carbon tetrachloride; however, little is known about its biological function. In this study, we examined the antimicrobial properties of pdtc and the mechanism of its antibiotic activity. The growth of
Pseudomonas stutzeri
strain KC, a pdtc-producing strain, was significantly enhanced by 32 μM pdtc. All nonpseudomonads and two strains of
P. stutzeri
were sensitive to 16 to 32 μM pdtc. In general, fluorescent pseudomonads were resistant to all concentrations tested. In competition experiments, strain KC demonstrated antagonism toward
Escherichia coli
. This effect was partially alleviated by 100 μM FeCl
3
. Less antagonism was observed in mutant derivatives of strain KC (CTN1 and KC657) which lack the ability to produce pdtc. A competitive advantage was restored to strain CTN1 by cosmid pT31, which restores pdtc production. pT31 also enhanced the pdtc resistance of all pdtc-sensitive strains, indicating that this plasmid contains elements responsible for resistance to pdtc. The antimicrobial effect of pdtc was reduced by the addition of Fe(III), Co(III), and Cu(II) and enhanced by Zn(II). Analyses by mass spectrometry determined that Cu(I):pdtc and Co(III):pdtc
2
form immediately under our experimental conditions. Our results suggest that pdtc is an antagonist and that metal sequestration is the primary mechanism of its antimicrobial activity. It is also possible that Zn(II), if present, may play a role in pdtc toxicity.
Biotransformation of Malachite Green by the FungusCunninghamella elegans
Authors: Cha Chang-Jun , Doerge Daniel R. , Cerniglia Carl E.
Published: July 2002

The filamentous fungusCunninghamella elegansATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 μmol h−1(mg of cells)−1. Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.
Evolution of the Lactic Acid Bacterial Community during Malt Whisky Fermentation: a Polyphasic Study
Authors: van Beek Sylvie , Priest Fergus G.
Published: July 2002

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to
Streptococcus
thermophilus
or
Saccharococcus
thermophilus
,
Lactobacillus
brevis
, and
Lactobacillus
fermentum
. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism.
Lactobacillus
casei
or
Lactobacillus
paracasei
,
L
.
fermentum
, and
Lactobacillus
ferintoshensis
were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to
Lactobacillus
acidophilus
and
Lactobacillus
delbrueckii
, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.
Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization
Authors: Ito Tsukasa , Nielsen Jeppe L. , Okabe Satoshi , Watanabe Yoshimasa , Nielsen Per H.
Published: July 2002

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that
Desulfobulbus
hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of
Desulfobulbus
cells detected with probe 660 could take up [
14
C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of
Desulfobulbus
cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H
2
-utilizing and
14
CO
2
-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H
2
-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the
Desulfomicrobium hypogeium
and the
Desulfovibrio desulfuricans
MB lineages, were found to be important H
2
utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.
Profiles of Airborne Fungi in Buildings and Outdoor Environments in the United States
Authors: Shelton Brian G. , Kirkland Kimberly H. , Flanders W. Dana , Morris George K.
Published: July 2002

We examined 12,026 fungal air samples (9,619 indoor samples and 2,407 outdoor samples) from 1,717 buildings located across the United States; these samples were collected during indoor air quality investigations performed from 1996 to 1998. For all buildings, both indoor and outdoor air samples were collected with an Andersen N6 sampler. The culturable airborne fungal concentrations in indoor air were lower than those in outdoor air. The fungal levels were highest in the fall and summer and lowest in the winter and spring. Geographically, the highest fungal levels were found in the Southwest, Far West, and Southeast. The most common culturable airborne fungi, both indoors and outdoors and in all seasons and regions, were
Cladosporium
,
Penicillium
, nonsporulating fungi, and
Aspergillus. Stachybotrys chartarum
was identified in the indoor air in 6% of the buildings studied and in the outdoor air of 1% of the buildings studied. This study provides industrial hygienists, allergists, and other public health practitioners with comparative information on common culturable airborne fungi in the United States. This is the largest study of airborne indoor and outdoor fungal species and concentrations conducted with a standardized protocol to date.
Quantitative Analysis of Cereulide, the Emetic Toxin of
Bacillus cereus
, Produced under Various Conditions
Authors: Häggblom Max M , Apetroaie Camelia , Andersson Maria A. , Salkinoja-Salonen Mirja S.
Published: July 2002

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by
Bacillus cereus.
The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml
−1
. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by
B. cereus
strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic
B. cereus
strains accumulated 80 to 166 μg of cereulide g
−1
(wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml
−1
in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10
8
to 6 × 10
8
CFU ml
−1
. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.
Species Diversity Improves the Efficiency of Mercury-Reducing Biofilms under Changing Environmental Conditions
Authors: von Canstein Harald , Kelly Sven , Li Ying , Wagner-Döbler Irene
Published: July 2002

Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant
Pseudomonas putida
strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater. The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency). Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped. In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater. Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations. The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis. Mercury-mediated selective pressure decreased the number of prevalent strains. Microbial diversity was completely restored after easing of the selective pressure. Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.
Transmission of Human T-Cell Lymphotropic Virus Type 1 Tax to Rabbits bytax-Only-Positive Human Cells
Authors: Zucker-Franklin Dorothea , Pancake Bette A. , Lalezari Parviz , Khorshidi Manoochehr
Published: July 2002

The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40taxin their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40tax. Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1taxsequences and antibodies to p40tax, while they lacked sequences and antibodies related togag,pol, orenv. Thus, it seemed important to determine whether the “tax-only” state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegativetax-only-positive MF patients or from healthytax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits becametaxsequence positive. These observations suggest that HTLV-1taxcan be transmitted bytax-only-positive mononuclear cells.
Treponema pallidumSurface Immunofluorescence Assay for Serologic Diagnosis of Syphilis
Authors: Marangoni Antonella , Sambri Vittorio , Storni Elisa , D'Antuono Antonietta , Negosanti Massimo , Cevenini Roberto
Published: July 2002

A surface immunofluorescence assay (SIFA) using live spirochetes was analyzed and compared with Western blot (WB), fluorescent treponemal antibody absorption (FTA-ABS), microhemagglutination (MHA-TP), andTreponema pallidumimmobilization (TPI) assays for detecting serum antibodies toT. pallidumin patients with syphilis, in disease controls, and in healthy subjects. SIFA and WB were 99% sensitive (99 of 100 positive specimens) and specific (140 of 140 negative specimens); FTA-ABS showed a sensitivity and a specificity of 90 and 89% (90 of 100 positive and 125 of 140 negative specimens), respectively. MHA-TP showed a sensitivity of 84% (84 of 100 positive specimens) and a specificity of 98.5% (138 of 140 negative specimens). Finally, TPI had a sensitivity of 52% (52 of 100 positive specimens) and a specificity of 100% (140 of 140 negative specimens). TheT. pallidumSIFA was therefore highly specific, showing no equivocal reactivities with control sera, and sensitive. The results suggest the possible use of SIFA as a confirmatory test in the serologic diagnosis of syphilis.
Development of an Enzyme-Linked Immunosorbent Assay for Serotyping
Ureaplasma urealyticum
Strains Using Monoclonal Antibodies
Authors: Echahidi Fedoua , Muyldermans Gaëtan , Lauwers Sabine , Naessens Anne
Published: July 2002

Ureaplasma urealyticum
comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of
U. urealyticum
isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains of
U. urealyticum
could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of
U. urealyticum
clinical isolates.
Effect of Protein SV-IV on ExperimentalSalmonella entericaSerovar Typhimurium Infection in Mice
Authors: Romano-Carratelli Caterina , Bentivoglio Concetta , Nuzzo Immacolata , Benedetto Nunzia , Buommino Elisabetta , Cozzolino Anna , Cartenì Maria , Morelli Francesco , Costanza Maria Rosaria , Metafora Biancamaria , Metafora Vittoria , Metafora Salvatore
Published: July 2002

Seminal vesicle protein IV (SV-IV) is a secretory anti-inflammatory, procoagulant, and immunomodulatory protein produced in large amounts by the seminal vesicle epithelium of the rat under the strict transcriptional control of androgen. In particular, this protein was shown to possess the ability to markedly inhibit in vivo the humoral and cell-mediated immune responses of mice to nonbacterial cellular antigens (sheep erythrocytes and spermatozoa). We report data that demonstrate that in mice treated with SV-IV and infected withSalmonella entericaserovar Typhimurium, SV-IV is able to downregulate some important immunological and biochemical parameters that serovar Typhimurium normally upregulates in these animals. This event did not correlate with a lower bacterial burden but was associated with a markedly increased one (300%). Furthermore, the treatment of mice with SV-IV alone also produced a significant increase in the rate of mortality among serovar Typhimurium-infected animals. The mechanism underlying these phenomena was investigated, and the strong immunosuppression produced by SV-IV in serovar Typhimurium-infected mice was suggested to be the basis for the increased rate of mortality. The SV-IV-mediated immunosuppression was characterized by a decrease in the humoral and cell-mediated immune responses, altered lymphocyte-macrophage interaction, downregulation of cytokine and inducible nitric oxide synthase gene expression, inhibition of macrophage phagocytosis and intracellular killing activities, and absence of apoptosis in the splenocyte population of SV-IV- and serovar Typhimurium-treated mice. The immunosuppressive activity of SV-IV was specific and was not due to aspecific cytotoxic effects. SV-IV-specific receptors (Kd= 10−8M) occurring on the macrophage and lymphocyte plasma membranes may be involved in the molecular mechanism underlying the SV-IV-mediated immunosuppression. Some results obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay also revealed a functional impairment of mitochondria (a decrease in mitochondrial dehydrogenase activity), thus indicating the possible implication of these organelles in the immunosuppressive process.
Activated T Lymphocytes Disappear from Circulation during Endotoxemia in Humans
Authors: Krabbe K. S. , Bruunsgaard H. , Qvist J. , Fonsmark L. , Møller K. , Hansen C. M. , Skinhøj P. , Pedersen B. K.
Published: July 2002

Seventeen volunteers received an intravenous bolus of endotoxin (2 ng/kg of body weight). Endotoxin-induced lymphopenia was constituted mainly by cells with an immature phenotype (CD45RA
+
CD45RO
−
) that were less likely to undergo apoptosis (CD28
+
), whereas cells with the highest rates of disappearance were characterized by an activated phenotype (CD45RA
−
CD45RO
+
) as well as a phenotype linked to apoptosis (CD95
+
CD28
−
). In conclusion, endotoxin-induced lymphopenia reflects the disappearance from the circulation of activated lymphocytes prone to undergo apoptosis.
Endocarditis Due to Rare and Fastidious Bacteria
Authors: Brouqui P. , Raoult D.
Published: July 2002

The etiologic diagnosis of infective endocarditis is easily made in the presence of continuous bacteremia with gram-positive cocci. However, the blood culture may contain a bacterium rarely associated with endocarditis, such as Lactobacillus spp., Klebsiella spp., or nontoxigenic Corynebacterium, Salmonella, Gemella, Campylobacter, Aeromonas, Yersinia, Nocardia, Pasteurella, Listeria, or Erysipelothrix spp., that requires further investigation to establish the relationship with endocarditis, or the blood culture may be uninformative despite a supportive clinical evaluation. In the latter case, the etiologic agents are either fastidious extracellular or intracellular bacteria. Fastidious extracellular bacteria such as Abiotrophia, HACEK group bacteria, Clostridium, Brucella, Legionella, Mycobacterium, and Bartonella spp. need supplemented media, prolonged incubation time, and special culture conditions. Intracellular bacteria such as Coxiella burnetii cannot be isolated routinely. The two most prevalent etiologic agents of culture-negative endocarditis are C. burnetti and Bartonella spp. Their diagnosis is usually carried out serologically. A systemic pathologic examination of excised heart valves including periodic acid-Schiff (PAS) staining and molecular methods has allowed the identification of Whipple's bacillus endocarditis. Pathologic examination of the valve using special staining, such as Warthin-Starry, Gimenez, and PAS, and broad-spectrum PCR should be performed systematically when no etiologic diagnosis is evident through routine laboratory evaluation.
Vaccinia Virus Inhibitors as a Paradigm for the Chemotherapy of Poxvirus Infections
Authors: De Clercq Erik
Published: July 2002

Poxviruses continue to pose a major threat to human health. Monkeypox is endemic in central Africa, and the discontinuation of the vaccination (with vaccinia virus) has rendered most humans vulnerable to variola virus, the etiologic agent of smallpox, should this virus be used in biological warfare or terrorism. However, a large variety of compounds have been described that are potent inhibitors of vaccinia virus replication and could be expected to be active against other poxviruses as well. These compounds could be grouped in different classes: (i) IMP dehydrogenase inhibitors (e.g., EICAR); (ii) SAH hydrolase inhibitors (e.g., 5′-noraristeromycin, 3-deazaneplanocin A, and various neplanocin A derivatives); (iii) OMP decarboxylase inhibitors (e.g., pyrazofurin) and CTP synthetase inhibitors (e.g., cyclopentenyl cytosine); (iv) thymidylate synthase inhibitors (e.g., 5-substituted 2′-deoxyuridines); (v) nucleoside analogues that are targeted at viral DNA synthesis (e.g., Ara-A); (vi) acyclic nucleoside phosphonates [e.g., (S)-HPMPA and (S)-HPMPC (cidofovir)]; and (vii) polyanionic substances (e.g., polyacrylic acid). All these compounds could be considered potential candidate drugs for the therapy and prophylaxis of poxvirus infections at large. Some of these compounds, in particular polyacrylic acid and cidofovir, were found to generate, on single-dose administration, a long-lasting protective efficacy against vaccinia virus infection in vivo. Cidofovir, which has been approved for the treatment of cytomegalovirus retinitis in immunocompromised patients, was also found to protect mice, again when given as a single dose, against a lethal aerosolized or intranasal cowpox virus challenge. In a biological warfare scenario, it would be advantageous to be able to use a single treatment for an individual exposed to an aerosolized poxvirus. Cidofovir thus holds great promise for treating human smallpox, monkeypox, and other poxvirus infections. Anecdotal experience points to the efficacy of cidofovir in the treatment of the poxvirus infections molluscum contagiosum and orf (ecthyma contagiosum) in immunosuppressed patients.
Identification of Mycobacterial Surface Proteins Released into Subcellular Compartments of Infected Macrophages
Authors: Beatty Wandy L. , Russell David G.
Published: July 2002

Considerable effort has focused on the identification of proteins secreted fromMycobacteriumspp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed.Mycobacterium bovisBCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.