bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Ciprofloxacin on Killing ofActinobacillus actinomycetemcomitansby Polymorphonuclear Leukocytes
Authors: Cacchillo David A. , Walters John D.
Published: July 2002

Actinobacillus actinomycetemcomitans, a pathogen associated with aggressive periodontitis, resists phagocytic killing by polymorphonuclear leukocytes (PMNs). It is susceptible to ciprofloxacin, which PMNs actively accumulate. This study tested the hypothesis that ciprofloxacin-loaded PMNs are more effective at killingA. actinomycetemcomitansthan control PMNs. Isolated human PMNs were loaded by brief incubation with 0.5 μg of ciprofloxacin/ml. Opsonized bacteria (ATCC 43718) were incubated at 37°C with control and ciprofloxacin-loaded PMNs and in the presence and absence of 0.5 μg of ciprofloxacin/ml. When assayed at bacteria-to-PMN ratios of 30:1 and 90:1, ciprofloxacin-loaded PMNs killed significantly more bacteria and achieved significantly shorter half times for killing than control PMNs (P< 0.05; Tukey's test). At ratios of 3:1 and 10:1, these differences were not significant.
Incorporation of Protease K into Larval Insect Membrane Vesicles Does Not Result in Disruption of Integrity or Function of the Pore-Forming
Bacillus thuringiensis
δ-Endotoxin
Authors: Aronson Arthur
Published: July 2002

Bacillus thuringiensis
δ-endotoxins insert into the brush border membranes of insect larval cells to form ion channels. A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin. There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores. These toxins, physically and functionally, appear to be confined to the membrane.
Use of Hydrostatic Pressure for Inactivation of Microbial Contaminants in Cheese
Authors: O'Reilly Ciara E. , O'Connor Paula M. , Kelly Alan L. , Beresford Thomas P. , Murphy Patrick M.
Published: July 2002

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (
Escherichia coli
K-12,
Staphylococcus aureus
ATCC 6538, and
Penicillium roqueforti
IMI 297987). Initially, cheese slurries inoculated with
E. coli
,
S. aureus
, and
P. roqueforti
were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30°C. At 400 MPa, the viability of
P. roqueforti
in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C.
S. aureus
and
E. coli
were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20°C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e.,
P. roqueforti
was more sensitive than
E. coli
, which was more sensitive than
S. aureus
. The organisms were more sensitive to pressure in cheese than slurry, especially with
E. coli
. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by
S. aureus
and
P. roqueforti
while greatest sensitivity to HP by
E. coli
was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.
Phylogenetic Characterization and In Situ Detection of a
Cytophaga-Flexibacter-Bacteroides
Phylogroup Bacterium in
Tuber borchii
Vittad. Ectomycorrhizal Mycelium
Authors: Barbieri Elena , Potenza Lucia , Rossi Ismaela , Sisti Davide , Giomaro Giovanna , Rossetti Simona , Beimfohr Claudia , Stocchi Vilberto
Published: July 2002

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus
Tuber borchii
Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of
T. borchii
Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each
T. borchii
mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the
Cytophaga-Flexibacter-Bacteroides
(CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus
T. borchii
was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the
Cytophaga-Flexibacter
phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus
Tuber
.
Evaluation of
p
-Naphtholbenzein-β-
d
-Galactoside as a Substrate for Bacterial β-Galactosidase
Authors: James A. L. , Chilvers K. F. , Perry J. D. , Armstrong L. , Gould F. K.
Published: July 2002

We describe the synthesis of a new substrate for the detection of β-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal) and cyclohexenoesculetinβ-
d
-galactoside (CHE-Gal). Of 206
Enterobacteriaceae
strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed
p
-naphtholbenzein-β-
d
-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of β-galactosidase.
Identification and Characterization of a Bile Acid 7α-Dehydroxylation Operon in
Clostridium
sp. Strain TO-931, a Highly Active 7α-Dehydroxylating Strain Isolated from Human Feces
Authors: Wells James E. , Hylemon Phillip B.
Published: July 2002

Clostridium
sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from
Clostridium
sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from
Eubacterium
sp. strain VPI 12708, another bile acid-7α-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from
Clostridium
sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the
Clostridium
sp. strain TO-931 operon are similar to those of a
Eubacterium
sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in the
Eubacterium
sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7α-dehydroxylation. In
Clostridium
sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3α-hydroxysteroid dehydrogenase, bile acid 7α-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from
Eubacterium
sp. strain VPI 12708 (encoded by
baiB
,
baiA
,
baiE
,
baiF
, and
baiG
, respectively). However, no genes similar to
Eubacterium
sp. strain VPI 12708
biaH
or
baiI
were found in the
Clostridium
sp. strain TO-931
bai
operon, and the two putative
Eubacterium
sp. strain VPI 12708 genes,
baiC
and
baiD
, were arranged in one continuous ORF in
Clostridium
sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a
bai
operator/promoter region identified in
Eubacterium
sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons of
Clostridium
sp. strain TO-931 and
Eubacterium
sp. strain VPI 12708 are highly conserved.
Nitrite Reductase Genes (
nirK
and
nirS
) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities
Authors: Braker Gesche , Zhou Jizhong , Wu Liyou , Devol Allan H. , Tiedje James M.
Published: July 2002

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying
nirK
and
nirS
genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria.
nirS
sequences could be amplified from samples of both sampling sites, whereas
nirK
sequences were detected only in samples from the Washington margin. To assess the underlying
nir
gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for
nirS
clones from Puget Sound and a slightly lower level of diversity for
nirK
and
nirS
clones from the Washington margin. One group dominated within
nirK
clones, but no dominance and only a few redundant clones were seen between sediment samples for
nirS
clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative
nirS
clones were
nirS
with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped
nirS
clones into three distinct subclusters within the
nirS
gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known
nirS
sequences or to isolates (mostly close relatives of
Pseudomonas stutzeri
) from the Washington margin sediment samples.
nirK
clones were more closely related to each other than were the
nirS
clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known
nirK
sequences. All
nirK
clones were also grouped into a distinct cluster which could not be placed with any strain with known
nirK
sequences. These findings show a very high diversity of
nir
sequences within small samples and that these novel
nir
clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.
Production of Exopolysaccharide by
Lactobacillus rhamnosus
R and Analysis of Its Enzymatic Degradation during Prolonged Fermentation
Authors: Pham P. L. , Dupont I. , Roy D. , Lapointe G. , Cerning J.
Published: July 2002

The potential of
Lactobacillus rhamnosus
R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by
L. rhamnosus
R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (
Y
EPS/S
) was 3.15 (0.0315 g of EPS [g of lactose]
−1
) and 2.88 (0.0288 g of EPS [g of glucose]
−1
). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (α-
d
-glucosidase, β-
d
-glucosidase, α-
d
-galactosidase, β-
d
-galactosidase, β-
d
-glucuronidase, and some traces of α-
l
-rhamnosidase). These enzymes were localized, two of them (α-
d
-glucosidase and β-
d
-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.
A Novel Protein-Deamidating Enzyme from
Chryseobacterium proteolyticum
sp. nov., a Newly Isolated Bacterium from Soil
Authors: Yamaguchi Shotaro , Yokoe Masaaki
Published: July 2002

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of
Chryseobacterium proteolyticum
strain 9670
T
isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus
Chryseobacterium
. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name
Chryseobacterium proteolyticum
is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).
Comparative Characterization of Complete and Truncated Forms of
Lactobacillus amylovorus
α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding
Authors: Rodriguez Sanoja R. , Morlon-Guyot J. , Jore J. , Pintado J. , Juge N. , Guyot J. P.
Published: July 2002

Two constructs derived from the α-amylase gene (
amyA
) of
Lactobacillus amylovorus
were expressed in
Lactobacillus plantarum
, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the

K
M

and
V
max
values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.
Growth of the Bacteriocin-Producing
Lactobacillus sakei
Strain CTC 494 in MRS Broth Is Strongly Reduced Due to Nutrient Exhaustion: a Nutrient Depletion Model for the Growth of Lactic Acid Bacteria
Authors: Leroy Frédéric , De Vuyst Luc
Published: July 2002

Although commercial MRS broth has been designed to allow excellent growth of lactobacilli, most of these bacteria are still subjected to a self-inhibiting process. The most likely explanation is the accumulation of lactic acid or other toxic end products and the depletion of nutrients. In this study, the self-inhibition of
Lactobacillus sakei
CTC 494 was analyzed in a kinetic way, and a nutrient depletion model was set up to describe the growth inhibition process. This simple model has considerable advantages compared to commonly used descriptive models such as the logistic growth equation. It offers a better fit and a more realistic description of the growth data by taking into account both growth inhibition due to lactic acid production and changes in growth rates due to nutrient depletion. Depending on the fermentation conditions, in MRS broth there appears to be a strong decrease of the specific growth rate over time. Some undefined compounds present in the complex nitrogen source of MRS broth appear to be of crucial importance because of their limited availability. Moreover, nutrient availability affects bacteriocin production through its effect on cell growth as well as on the bacteriocin production per cell. A plateau value for the bacteriocin production by
L. sakei
CTC 494 was observed.
New Additive for Culture Media for Rapid Identification of Aflatoxin-Producing
Aspergillus
Strains
Authors: Fente C. A. , Ordaz J. Jaimez , Vázquez B. I. , Franco C. M. , Cepeda A.
Published: July 2002

A new reliable, fast, and simple method for the detection of aflatoxigenic
Aspergillus
strains, consisting of the addition of a cyclodextrin (a methylated β-cyclodextrin derivative) to common media used for testing mycotoxin production ability, was developed. We propose the use of this compound as an additive for fungal culture media to enhance the natural fluorescence of aflatoxins. The production of aflatoxins coincided with the presence of a bright blue or blue-green fluorescent area surrounding colonies when observed under long-wavelength (365-nm) UV light after 3 days of incubation at 28°C. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by high-pressure liquid chromatography with fluorescence detection.
Improved Method for Detection of
Vibrio parahaemolyticus
in Seafood
Authors: Hara-Kudo Yukiko , Nishina Tokuhiro , Nakagawa Hiroshi , Konuma Hirotaka , Hasegawa Junko , Kumagai Susumu
Published: July 2002

We have developed a new, effective procedure for detecting
Vibrio parahaemolyticus
in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for
V. parahaemolyticus
. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The
V. parahaemolyticus
colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains.
V. parahaemolyticus
was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of
V. parahaemolyticus
. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying
V. parahaemolyticus
in seafood samples than previously used methods.
Efficient Transformation System for
Propionibacterium freudenreichii
Based on a Novel Vector
Authors: Jore J. P. M. , van Luijk N. , Luiten R. G. M. , van der Werf M. J. , Pouwels P. H.
Published: July 2002

A 3.6-kb endogenous plasmid was isolated from a
Propionibacterium freudenreichii
strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from
Mycobacterium
, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an
Escherichia coli
-specific replicon and an erythromycin resistance gene for selection in
Propionibacterium
. Hybrid vectors obtained in this way replicated in both
E. coli
and
P. freudenreichii
. Whereas electroporation of
P. freudenreichii
with vector DNA isolated from an
E. coli
transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a
Propionibacterium
transformant dramatically increased the efficiency of transformation (≥10
8
colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of
Propionibacterium
with DNA ligation mixtures.
Microorganisms with a Taste for Vanilla: Microbial Ecology of Traditional Indonesian Vanilla Curing
Authors: Röling Wilfred F. M. , Kerler Josef , Braster Martin , Apriyantono Anton , Stam Hein , van Verseveld Henk W.
Published: July 2002

The microbial ecology of traditional postharvesting processing of vanilla beans (curing) was examined using a polyphasic approach consisting of conventional cultivation, substrate utilization-based and molecular identification of isolates, and cultivation-independent community profiling by 16S ribosomal DNA based PCR-denaturing gradient gel electrophoresis. At two different locations, a batch of curing beans was monitored. In both batches a major shift in microbial communities occurred after short-term scalding of the beans in hot water. Fungi and yeast disappeared, although regrowth of fungi occurred in one batch during a period in which process conditions were temporarily not optimal. Conventional plating showed that microbial communities consisting of thermophilic and thermotolerant bacilli (mainly closely related to
Bacillus subtilis, B. licheniformis,
, and
B. smithii
) developed under the high temperatures (up to 65°C) that were maintained for over a week after scalding. Only small changes in the communities of culturable bacteria occurred after this period. Molecular analysis revealed that a proportion of the microbial communities could not be cultured on conventional agar medium, especially during the high-temperature period. Large differences between both batches were observed in the numbers of microorganisms, in species composition, and in the enzymatic abilities of isolated bacteria. These large differences indicate that the effects of microbial activities on the development of vanilla flavor could be different for each batch of cured vanilla beans.
Quorum Sensing in the Dimorphic Fungus
Candida albicans
Is Mediated by Farnesol
Authors: Hornby Jacob M. , Jensen Ellen C. , Lisec Amber D. , Tasto Joseph J. , Jahnke Brandon , Shoemaker Richard , Dussault Patrick , Nickerson Kenneth W.
Published: July 2002

The inoculum size effect in the dimorphic fungus
Candida albicans
results from production of an extracellular quorum-sensing molecule (QSM). This molecule prevents mycelial development in both a growth morphology assay and a differentiation assay using three chemically distinct triggers for germ tube formation (GTF):
l
-proline,
N
-acetylglucosamine, and serum (either pig or fetal bovine). In all cases, the presence of QSM prevents the yeast-to-mycelium conversion, resulting in actively budding yeasts without influencing cellular growth rates. QSM exhibits general cross-reactivity within
C. albicans
in that supernatants from strain A72 are active on five other strains of
C. albicans
and vice versa. The QSM excreted by
C. albicans
is farnesol (C
15
H
26
O; molecular weight, 222.37). QSM is extracellular, and is produced continuously during growth and over a temperature range from 23 to 43°C, in amounts roughly proportional to the CFU/milliliter. Production is not dependent on the type of carbon source nor nitrogen source or on the chemical nature of the growth medium. Both commercial mixed isomer and (
E,E
)-farnesol exhibited QSM activity (the ability to prevent GTF) at a level sufficient to account for all the QSM activity present in
C. albicans
supernatants, i.e., 50% GTF at ca. 30 to 35 μM. Nerolidol was ca. two times less active than farnesol. Neither geraniol (C
10
), geranylgeraniol (C
20
), nor farnesyl pyrophosphate had any QSM activity.
Characterization of the 13-Kilobase
ermF
Region of the
Bacteroides
Conjugative Transposon CTnDOT
Authors: Whittle Gabrielle , Hund Bonnie D. , Shoemaker Nadja B. , Salyers Abigail A.
Published: July 2002

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an
ermF
gene that is not found on CTnERL. In this report, we show that the region containing
ermF
appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the
ermF
region contains genes also carried on
Bacteroides
transposons Tn
4351
and Tn
4551
, it does not contain the IS
4351
element which is found on these transposons. In CTnDOT, insertion of the
ermF
region occurred near a stem-loop structure at the end of
orf2
, an open reading frame located immediately downstream of the integrase (
int
) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the
ermF
region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the
Bacteroides
MTn Tn
4399
. Analysis of 19 independent
Bacteroides
isolates showed that the
ermF
region is located in the same position in all of the strains analyzed and that the compositions of the
ermF
region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of
Bacteroides
were derived from a common ancestor and proliferated in the diverse
Bacteroides
population.
The Phosphinomethylmalate Isomerase Gene
pmi,
Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide in
Streptomyces viridochromogenes
Authors: Heinzelmann E. , Kienzlen G. , Kaspar S. , Recktenwald J. , Wohlleben W. , Schwartz D.
Published: July 2002

Streptomyces viridochromogenes
Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222,
in
L. C. Vining and C. Stuttard, ed.,
Genetics and Biochemistry of Antibiotic Production,
1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene,
pmi
) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the
S. viridochromogenes
TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a
pmi
mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in
Streptomyces lividans
. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an
acnA
mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.
Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity
Authors: Guder André , Schmitter Tim , Wiedemann Imke , Sahl Hans-Georg , Bierbaum Gabriele
Published: July 2002

The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids which inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II and which is produced by
Bacillus
sp. strain HIL Y-85,54728. The structural gene of mersacidin as well as accessory genes is organized in a biosynthetic gene cluster which is located on the chromosome and contains three open reading frames with similarities to regulatory proteins:
mrsR2
and
mrsK2
encode two proteins with homology to bacterial two-component systems, and
mrsR1
shows similarity to a response regulator. Both
mrsR2/K2
and
mrsR1
were inactivated by insertion of an antibiotic resistance marker. Disruption of
mrsR1
resulted in loss of mersacidin production; however, producer self-protection was not impaired. In contrast, inactivation of
mrsR2/K2
led to an increased susceptibility to mersacidin whereas biosynthesis of the lantibiotic remained unaffected. Binding of mersacidin to intact cells was significantly enhanced in the
mrsR2/K2
knockout mutant. Reverse transcription-PCR analysis from total RNA preparations showed that in contrast to the wild-type strain, the structural genes of the ABC transporter MrsFGE were not transcribed in the knockout mutant. It was therefore concluded that producer self-protection against mersacidin is conferred by the ABC transporter MrsFGE and that the transcription of
mrsFGE
is regulated by MrsR2/K2, whereas production of the antibacterial peptide is solely activated by MrsR1.
Engineering of a
Bacillus subtilis
Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin
Authors: Wu Sau-Ching , Wong Sui-Lam
Published: July 2002

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the
Bacillus subtilis
expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using
B. subtilis
face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a
B. subtilis
strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the
B. subtilis
chromosomal biotin biosynthetic operon (
bioWAFDBIorf2
) with an engineered one consisting of the
B. subtilis groE
promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.
Shedding ofEscherichia coliO157:H7 in Dairy Cattle Housed in a Confined Environment following Waterborne Inoculation
Authors: Shere J. A. , Kaspar C. W. , Bartlett K. J. , Linden S. E. , Norell B. , Francey S. , Schaefer D. M.
Published: July 2002

A study ofEscherichia coliO157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration ofE. coliO157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers ofE. coliO157:H7 isolates shed in these calves varied from 60 to 105CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by usingXbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 103to 104CFU ofE. coliO157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 102to 106CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 106CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination ofE. coliO157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.
Enrichment of Members of the Family
Geobacteraceae
Associated with Stimulation of Dissimilatory Metal Reduction in Uranium-Contaminated Aquifer Sediments
Authors: Holmes Dawn E. , Finneran Kevin T. , O'Neil Regina A. , Lovley Derek R.
Published: July 2002

Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family
Geobacteraceae
, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion,
Geobacteraceae
accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas
Geobacteraceae
accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these
Geobacteraceae
sequences were most similar to sequences from
Desulfuromonas
species, with the remainder being most closely related to
Geobacter
species. Quantitative analysis of
Geobacteraceae
sequences with most-probable-number PCR and TaqMan analyses indicated that the number of
Geobacteraceae
sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in
Geobacteraceae
sequences was observed in control sediments. In contrast to the predominance of
Geobacteraceae
sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that
Geobacteraceae
are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that
Geobacteraceae
will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.
Biodegradation of Methyl
tert
-Butyl Ether and Other Fuel Oxygenates by a New Strain,
Mycobacterium austroafricanum
IFP 2012
Authors: François Alan , Mathis Hugues , Godefroy Davy , Piveteau Pascal , Fayolle Françoise , Monot Frédéric
Published: July 2002

A strain that efficiently degraded methyl
tert
-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound
tert
-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as
Mycobacterium austroafricanum
IFP 2012, was also able to degrade
tert
-amyl methyl ether and
tert
-amyl alcohol. Ethyl
tert
-butyl ether was weakly degraded.
tert
-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co
2+
during growth of
M. austroafricanum
IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.
A Strain ofBacillus sphaericusCauses Slower Development of Resistance inCulex quinquefasciatus
Authors: Pei Guofeng , Oliveira Cláudia M. F. , Yuan Zhiming , Nielsen-LeRoux Christina , Silva-Filha Maria Helena , Yan Jianpin , Regis Lêda
Published: July 2002

Two field-collectedCulex quinquefasciatuscolonies were subjected to selection pressure by three strains ofBacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection withB. sphaericusC3-41 and 2362 showed very high levels of cross-resistance toB. sphaericus2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all theB. sphaericus-selected colonies showed no cross-resistance toBacillus thuringiensissubsp.israelensis, suggesting that it would be a promising alternative in managing resistance toB. sphaericusinC. quinquefasciatuslarvae.
Ageing Is Associated with a Prolonged Fever Response in Human Endotoxemia
Authors: Krabbe Karen Suárez , Bruunsgaard Helle , Hansen Christian Muff , Møller Kirsten , Fonsmark Lise , Qvist Jesper , Madsen Per Lav , Kronborg Gitte , Andersen Henrik Ørbaek , Skinhøj Peter , Pedersen Bente Klarlund
Published: July 2002

The purpose of this study was to investigate whether an age-associated impaired acute-phase response exists. Nine healthy elderly volunteers (median, 66 years; range, 61 to 69 years) and eight young controls (median, 24 years; range, 20 to 27 years) were given an intravenous bolus of endotoxin (2 ng/kg). The rectal temperature was monitored continuously, and blood samples for cytokine measurements were obtained before endotoxin administration as well as 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 24 h after the injection. The elderly subjects showed a more prolonged fever response compared to the young controls. Levels of tumor necrosis factor alpha (TNF-α), soluble TNF receptors (sTNFR-I), interleukin-6 (IL-6), IL-8, IL-10, and IL-1 receptor antagonist (IL-1ra) in plasma increased markedly following endotoxin administration in both groups. The elderly group showed larger initial increases in TNF-α and sTNFR-I levels and prolonged increased levels of sTNFR-I. Monocyte concentrations decreased in both groups, with the elderly group showing a more rapid decrease and a slower subsequent increase than did the young group. Furthermore, the elderly group had a more rapid increase in C-reactive protein levels than did the young group. In conclusion, ageing is associated with an altered acute-phase response including initial hyperreactivity, prolonged inflammatory activity, and prolonged fever response.
Treatment with Neurokinin-1 Receptor Antagonist Reduces Severity of Inflammatory Bowel Disease Induced byCryptosporidium parvum
Authors: Sonea Ioana M. , Palmer Mitchell V. , Akili Dhuha , Harp James A.
Published: July 2002

Inflammatory bowel disease (IBD) is a chronic, debilitating disorder of uncertain and perhaps multiple etiologies. It is believed to be due in part to disregulation of the immune system. Neuroimmune interactions may be involved in induction or maintenance of IBD. In the present study, we examined the potential role of a neurotransmitter, substance P, in a mouse model of IBD. We found that binding sites for substance P, and more specifically, neurokinin-1 receptors, were upregulated in intestinal tissue of mice with IBD-like syndrome. Dosing of mice with LY303870, a neurokinin-1 receptor antagonist, reduced the severity of IBD, and treatment of mice with preexisting IBD allowed partial healing of lesions. We hypothesize that blocking the binding of substance P to the neurokinin-1 receptor interrupts the inflammatory cascade that triggers and maintains intestinal lesions of IBD.
Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa
Authors: Upcroft Peter , Upcroft Jacqueline A.
Published: July 2002

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.
Gallium Disrupts Iron Metabolism of Mycobacteria Residing within Human Macrophages
Authors: Olakanmi Oyebode , Britigan Bradley E. , Schlesinger Larry S.
Published: July 2002

Mycobacterium tuberculosisandM. aviumcomplex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of bothM. tuberculosisand MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga3+) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe3+and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO3)3and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition ofM. tuberculosisstrains and MAC grown extracellularly and within human macrophages. Ga is bactericidal forM. tuberculosisgrowing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomalM. tuberculosisand that Ga inhibits this Fe acquisition. Thus, Ga(NO3)3disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.