bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemostat Enrichments of Human Feces with Resistant Starch Are Selective for Adherent Butyrate-Producing Clostridia at High Dilution Rates
Authors: Sharp Richard , Macfarlane George T.
Published: July 2002

Resistant starch (RS) enrichments were made using chemostats inoculated with human feces from two individuals at two dilution rates (
D
= 0.03 h
−1
and
D
= 0.30 h
−1
) to select for slow- and fast-growing amylolytic communities. The fermentations were studied by analysis of short-chain fatty acids, amylase and α-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide probes. Considerable butyrate was produced at
D
= 0.30 h
−1
, which corresponded with reduced branched-chain fatty acid formation. At both dilution rates, high levels of extracellular amylase activity were produced, while α-glucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia became more important at
D
= 0.30 h
−1
. Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominated in RS enrichments at
D
= 0.30 h
−1
. This organism attached apically to RS granules and formed rosette-like structures which, with glycocalyx formation, agglomerated to form biofilm networks in the planktonic phase. Attempts to isolate this bacterium in pure culture were repeatedly unsuccessful, although a single colony was eventually obtained. On the basis of its 16S rDNA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I
Clostridium
sp. A 16S rRNA-targeted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at
D
= 0.03 and 0.30 h
−1
, respectively. This study indicates that bacterial populations with significant metabolic potential can be overlooked using culture-based methodologies. This may provide a paradigm for explaining the discrepancy between the low numbers of butyrate-producing bacteria that are isolated from fecal samples and the actual production of butyrate.
A Fruiting Body Tissue Method for Efficient
Agrobacterium
-Mediated Transformation of
Agaricus bisporus
Authors: Chen Xi , Stone Michelle , Schlagnhaufer Carl , Romaine C. Peter
Published: July 2002

We describe a modified
Agrobacterium
-mediated method for the efficient transformation of
Agaricus bisporus
. Salient features of this procedure include cocultivation of
Agrobacterium
and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.
Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from
Brevibacterium helvolum
Authors: Kim Yong Hwan , Kwon Tae Keun , Park Sungsoon , Seo Hak Soo , Cheong Jong-Joo , Kim Chung Ho , Kim Ju-Kon , Lee Jong Seob , Choi Yang Do
Published: July 2002

A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium
Brevibacterium helvolum
. The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides. Recombinant
BvMTS
,
BvMTH
, and fusion gene
BvMTSH
, constructed by insertion of an adenylate in the overlapping region, were expressed in
Escherichia coli
. Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose. The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose. Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units. The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes. The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production. Addition of α-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.
Persistent Colonization of Sheep byEscherichia coliO157:H7 and OtherE. coliPathotypes
Authors: Cornick N. A. , Booher S. L. , Casey T. A. , Moon H. W.
Published: July 2002

Shiga toxin-producingEscherichia coli(STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep byE. coliO157:H7 to that by other pathotypes ofE. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains ofE. coliO157:H7, two strains of enterotoxigenicE. coli(ETEC), and one strain of enteropathogenicE. coli. Both STEC strains and ETEC 2041 were given at either 107or 1010CFU/strain/animal. The other strains were given only at 1010CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 107or 1010CFU. One of the ETEC strains also persisted when inoculated at 1010CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 107CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes ofE. coli.
Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides
Authors: Deutsch Stéphanie-Marie , Molle Daniel , Gagnaire Valérie , Piot Michel , Atlan Danièle , Lortal Sylvie
Published: July 2002

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from
Lactobacillus helveticus
ITG LH1, ITG LH77, and CNRZ 32,
Lactobacillus delbrueckii
subsp.
lactis
ITG LL14 and ITG LL51,
L. delbrueckii
subsp.
bulgaricus
CNRZ 397 and NCDO 1489, and
Streptococcus thermophilus
CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain,
L. helveticus
was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of
S. thermophilus
were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.
Effect of Calcium in Assay Medium on
D
Value of
Bacillus stearothermophilus
ATCC 7953 Spores
Authors: Sasaki Koichi , Shintani Hideharu , Itoh Junpei , Kamogawa Takuji , Kajihara Yousei
Published: July 2002

The
D
value of commercial biological indicator spore strips using
Bacillus stearothermophilus
ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of
D
value.
Selective Removal of Nitrogen from Quinoline and Petroleum by
Pseudomonas ayucida
IGTN9m
Authors: Kilbane John J. , Ranganathan Rajaram , Cleveland Lisa , Kayser Kevin J. , Ribiero Claudia , Linhares Monica M.
Published: July 2002

Enrichment culture experiments employing soil and water samples obtained from petroleum-contaminated environments succeeded in the isolation of a pure culture possessing the ability to utilize quinoline as a sole nitrogen source but did not utilize quinoline as a carbon source. This culture was identified as
Pseudomonas ayucida
based on a partial 16S rRNA gene sequence, and the strain was given the designation IGTN9m. Examination of metabolites using thin-layer chromatography and gas chromatography-mass spectrometry suggests that
P. ayucida
IGTN9m converts quinoline to 2-quinolinone and subsequently to 8-hydroxycoumarin. Resting cells of
P. ayucida
IGTN9m were shown to be capable of selectively removing about 68% of quinoline from shale oil in a 16-h treatment time. These results suggest that
P. ayucida
IGTN9m may be useful in petroleum biorefining for the selective removal of organically bound nitrogen from petroleum.
Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation
Authors: Crocetti Gregory R. , Hugenholtz Philip , Bond Philip L. , Schuler Andrew , Keller Jürg , Jenkins David , Blackall Linda L.
Published: July 2002

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2
Proteobacteria
arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the
Actinobacteria
. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2
Proteobacteria
were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to
Rhodocyclus
spp. (94 to 97% identity) and
Propionibacter pelophilus
(95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2
Proteobacteria
probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to
Rhodocyclus
and
Propionibacter
.
Comparative Experiments To Examine the Effects of Heating on Vegetative Cells and Spores ofClostridium perfringensIsolates Carrying Plasmid Genes versus Chromosomal Enterotoxin Genes
Authors: Sarker Mahfuzur R. , Shivers Robert P. , Sparks Shauna G. , Juneja Vijay K. , McClane Bruce A.
Published: July 2002

Clostridium perfringensenterotoxin (CPE) is an important virulence factor for bothC. perfringenstype A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated thatC. perfringensisolates associated with food poisoning carry a chromosomalcpegene, while non-food-borne human gastrointestinal disease isolates carry a plasmidcpegene. However, no explanation has been provided for the strong associations between certaincpegenotypes and particular CPE-associated diseases. SinceC. perfringensfood poisoning usually involves cooked meat products, we hypothesized that chromosomalcpeisolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmidcpeisolates, (ii) heating induces loss of thecpeplasmid, or (iii) heating induces migration of the plasmidcpegene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomalcpeisolates were found to exhibit, on average approximately twofold-higher decimal reduction values (Dvalues) at 55°C than vegetative cells of plasmidcpeisolates exhibited. Furthermore, the spores of chromosomalcpeisolates had, on average, approximately 60-fold-higherDvalues at 100°C than the spores of plasmidcpeisolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained theircpegene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomalcpeisolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.
Detection of Oxytetracycline Production byStreptomyces rimosusin Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry
Authors: Hansen Lars Hestbjerg , Ferrari Belinda , Sørensen Anders Hay , Veal Duncan , Sørensen Søren Johannes
Published: July 2002

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production byStreptomyces rimosusin soil microcosms. A plasmid containing a transcriptional fusion between thetetR-regulated Ptetpromoter from Tn10and a FACS-optimizedgfpgene was constructed. When harbored byEscherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline byS. rimosusintroduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.
Physiological Role of β-Phosphoglucomutase in
Lactococcus lactis
Authors: Levander Fredrik , Andersson Ulrika , Rådström Peter
Published: July 2002

A β-phosphoglucomutase (β-PGM) mutant of
Lactococcus lactis
subsp.
lactis
ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h
−1
, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h
−1
on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in
L. lactis
.
Iron-Binding Compounds from
Agrobacterium
spp.: Biological Control Strain
Agrobacterium rhizogenes
K84 Produces a Hydroxamate Siderophore
Authors: Penyalver Ramón , Oger Philippe , López Marı́a M. , Farrand Stephen K.
Published: July 2002

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of
Agrobacterium
strains belonging to the species
A. tumefaciens, A. rhizogenes
, and
A. vitis
. The crown gall biocontrol agent
A. rhizogenes
strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn
5
-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn
5
insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn
5
was located in the mutant. Sequence analysis showed that the Tn
5
insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of
A. rhizogenes
that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.
Nisin Resistance of
Streptococcus bovis
Authors: Mantovani Hilário C. , Russell James B.
Published: July 2002

The growth of
Streptococcus bovis
JB1 was initially inhibited by nisin (1 μM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 μM nisin was added, but resistant cells retained potassium even after addition of 10 μM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome
c
, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of
S. bovis
to nisin.
S. bovis
JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.
Determination of DNA Content of Aquatic Bacteria by Flow Cytometry
Authors: Button D. K. , Robertson Betsy R.
Published: July 2002

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a
Marinobacter arcticus
isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell
−1
was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell
−1
.
Detection of the Bacteriocin Propionicin PLG-1 with Polyvalent Anti-PLG-1 Antiserum
Authors: Leversee Jay A. , Glatz Bonita A.
Published: July 2002

Polyclonal antibodies against the bacteriocin propionicin PLG-1 were produced in rabbits at high titer (256,000 to 512,000, as determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain
Propionibacterium thoenii
P127 within the first 24 h of incubation, but bacteriocin activity was not detected in the same culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect ELISA at 5 and 12 days of incubation for production of cross-reacting protein and by well diffusion assay for bacteriocin activity. Cross-reacting protein was detected in 52 strains: all of the tested strains of
P. thoenii
, most of the strains of
Propionibacterium jensenii
, and a minority of the
Propionibacterium acidipropionici
and
Propionibacterium freudenreichii
strains. Of these 52 strains, only 4 strains of
P. thoenii
showed bacteriocin activity in a well diffusion assay. Eight bacteriocin-negative mutants of strain P127 were negative in both ELISA and well diffusion assays. Western blot analysis showed that three protein bands bound anti-PLG-1 antibodies in culture supernatants: a 9.1-kDa band that is assumed to be the PLG-1 monomer and 16.2- and 27.5-kDa bands that may be precursors, multimers, or complexes of PLG-1.
Identification of Deoxynivalenol- and Nivalenol-Producing Chemotypes of
Gibberella zeae
by Using PCR
Authors: Lee Theresa , Oh Dae-Woong , Kim Hye-Seon , Lee Jungkwan , Kim Yong-Ho , Yun Sung-Hwan , Lee Yin-Won
Published: July 2002

Gibberella zeae
, a major cause of cereal scab, may be divided into two chemotypes based on production of the trichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced the gene cluster for trichothecene biosynthesis from each chemotype.
G. zeae
H-11 is a DON producer isolated from corn, and
G. zeae
88-1 is a NIV producer from barley. We sequenced a 23-kb gene cluster from H-11 and a 26-kb cluster from 88-1, along with the unlinked
Tri101
genes. Each gene cluster contained 10
Tri
gene homologues in the same order and transcriptional directions as those of
Fusarium sporotrichioides
. Between H-11 and 88-1 all of the
Tri
homologues except
Tri7
were conserved, with identities ranging from 88 to 98% and 82 to 99% at the nucleotide and amino acid levels, respectively. The
Tri7
sequences were only 80% identical at the nucleotide level. We aligned the
Tri7
genes and found that the
Tri7
open reading frame of H-11 carried several mutations and an insertion containing 10 copies of an 11-bp tandem repeat. The
Tri7
gene from 88-1 carried neither the repeat nor the mutations. We assayed 100
G. zeae
isolates of both chemotypes by PCR amplification with a primer pair derived from the
Tri7
gene and could differentiate the chemotypes by polyacrylamide gel electrophoresis. The PCR-based method developed in this study should provide a simple and reliable diagnostic tool for differentiating the two chemotypes of
G. zeae
.
Degradation of 3-Methylpyridine and 3-Ethylpyridine by
Gordonia nitida
LE31
Authors: Lee Jay J. , Rhee Sung-Keun , Lee Sung-Taik
Published: July 2002

Cells of
Gordonia nitida
LE31 grown on 3-methylpyridine degraded 3-ethylpyridine without a lag time and vice versa. Cyclic intermediates were not detected, but formic acid was identified as a metabolite. Degradation of levulinic acid was induced in cells grown on 3-methylpyridine and 3-ethylpyridine. Levulinic aldehyde dehydrogenase and formamidase activities were higher in cells grown on 3-methylpyridine and 3-ethylpyridine than in cells grown on acetate. These data indicate that 3-methylpyridine and 3-ethylpyridine were degraded via a new pathway involving C-2–C-3 ring cleavage.
Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors
Authors: Heuer Holger , Kroppenstedt Reiner M. , Lottmann Jana , Berg Gabriele , Smalla Kornelia
Published: July 2002

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for
Bacteria
,
Actinomycetales
, or α- or β-
Proteobacteria
. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.
Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods
Authors: Leclercq A. , Wanegue C. , Baylac P.
Published: July 2002

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as
Hafnia alvei
, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed
Escherichia coli
in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (
P
> 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were
E. coli
and
H. alvei
, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by
E. coli
enumeration to estimate food sanitary quality should be recommended.
Siderophore Typing, a Powerful Tool for the Identification of Fluorescent and Nonfluorescent Pseudomonads
Authors: Meyer Jean-Marie , Geoffroy Valérie A. , Baida Nader , Gardan Louis , Izard Daniel , Lemanceau Philippe , Achouak Wafa , Palleroni Norberto J.
Published: July 2002

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species
Pseudomonas brassicacearum
,
Pseudomonas fuscovaginae
,
Pseudomonas jessenii
,
Pseudomonas mandelii
,
Pseudomonas monteilii
, “
Pseudomonas mosselii
,” “
Pseudomonas palleronii
,”
Pseudomonas rhodesiae
, “
Pseudomonas salomonii
,”
Pseudomonas syringae
,
Pseudomonas thivervalensis
,
Pseudomonas tolaasii
, and
Pseudomonas veronii
and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous
Pseudomonas fluorescens
species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species
Pseudomonas corrugata
,
Pseudomonas frederiksbergensis
,
Pseudomonas graminis
, and
Pseudomonas plecoglossicida
, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.
Enzyme-Linked Immunosorbent Assays Using the Recombinant Dense Granule Antigens GRA6 and GRA1 of
Toxoplasma gondii
for Detection of Immunoglobulin G Antibodies
Authors: Lecordier Laurence , Fourmaux Marie-Pierre , Mercier Corinne , Dehecq Eric , Masy E. , Cesbron-Delauw Marie-France
Published: July 2002

The potential of the dense granule antigens GRA1 and GRA6 of
Toxoplasma gondii
to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed in
Escherichia coli
as glutathione-
S
-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred
T. gondii
-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.
An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of
Cryptosporidium
Oocysts
Authors: Weir C. , Vesey G. , Slade M. , Ferrari B. , Veal D. A. , Williams K.
Published: July 2002

The detection of
Cryptosporidium
oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to
Cryptosporidium
oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of
Cryptosporidium
oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG
1
monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.
High Prevalence of
Helicobacter pylori
in the Alaska Native Population and Association with Low Serum Ferritin Levels in Young Adults
Authors: Parkinson Alan J. , Gold Benjamin D. , Bulkow Lisa , Wainwright Robert B. , Swaminathan Balasubra , Khanna Bhawna , Petersen Kenneth M. , Fitzgerald Mary Anne
Published: July 2002

Iron deficiency anemia is a common public health problem in the Alaska Native population. Yet, a clear etiology has eluded researchers for decades. Previous studies suggested a link between
Helicobacter pylori
infection, gastrointestinal blood loss due to hemorrhagic gastritis, and generalized iron deficiency anemia in adult Alaska Natives. Therefore, we examined the association between the prevalence of
H. pylori
-specific immunoglobulin G (IgG) and serum ferritin levels, a marker of iron deficiency. A random sample of 2,080 serum samples from Alaska Native residents drawn between 1980 and 1986 from residents in 13 regions was selected, and the samples were stratified by age, sex, and region. Overall, 75% were positive for
H. pylori
-specific IgG. The rate of
H. pylori
seropositivity increased with age; by age 14 years, 78% of the residents were positive. There were no gender differences in
H. pylori
seropositivity. However, marked regional differences were observed. Serum ferritin levels of <12 ng/ml were found most commonly among persons <20 years of age and among women of childbearing age. A significant association between low serum ferritin levels and prevalence of
H. pylori
-specific IgG was found, particularly for people aged less than 20 years.
H. pylori
may be a factor contributing to the iron deficiency anemia in the Alaska Native population.
Presence of Human T-Cell Responses to theMycobacterium leprae45-Kilodalton Antigen Reflects Infection with or Exposure toM. leprae
Authors: Macfarlane Anne , Mondragon-Gonzalez Rafael , Vega-Lopez Francisco , Wieles Brigitte , de Pena Josefina , Rodriguez Obdulia , Suarez y de la Torre Raul , de Vries Rene R. P. , Ottenhoff Tom H. M. , Dockrell Hazel M.
Published: July 2002

The ability of the 45-kDa serine-richMycobacterium lepraeantigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to theM. leprae45-kDa antigen (92.8%;n= 14). Responses were lower in lepromatous leprosy patients (60.6%;n= 34), but some responses to the 45-kDa antigen were detected in patients unresponsive toM. lepraesonicate. The proportion of positive responses to theM. leprae45-kDa antigen was much higher in leprosy contacts (88%;n= 17) than in controls from areas of endemicity (10%;n= 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the nativeMycobacterium tuberculosis30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that theM. leprae45-kDa antigen is a potent T-cell antigen which isM. lepraespecific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.
groEL
Encodes a Highly Antigenic Protein in
Burkholderia pseudomallei
Authors: Woo Patrick C. Y. , Leung Patricia K. L. , Wong Samson S. Y. , Ho Pak-Leung , Yuen Kwok-Yung
Published: July 2002

No recombinant protein is available for serodiagnosis of melioidosis. In this study, we report the cloning of the
groEL
gene, which encodes an immunogenic protein of
Burkholderia pseudomallei
. Bidirectional DNA sequencing of
groEL
revealed that the gene contained a single open reading frame encoding 546 amino acid residues with a predicted molecular mass of 57.1 kDa. Basic Local Alignment Search Tool analysis showed that the putative protein encoded by
groEL
is homologous to the chaperonins encoded by the
groEL
genes of other bacteria. It has 98% amino acid identity with the GroEL of
Burkholderia cepacia,
98% amino acid identity with the GroEL of
Burkholderia vietnamiensis,
and 82% amino acid identity with the GroEL of
Bordetella pertussis
. Furthermore, it was observed that patients with melioidosis develop a strong antibody response against GroEL, suggesting that the recombinant protein and its monoclonal antibody may be useful for serodiagnosis in patients with melioidosis and that the protein may represent a good cell surface target for host humoral immunity. Further studies in these directions would be warranted.
Recognition of Multiple Classes of Hepatitis C Antibodies Increases Detection Sensitivity in Oral Fluid
Authors: Zmuda Jonathan F. , Wagoneer Barbara , Liotta Lance , Whiteley Gordon
Published: July 2002

Paired serum-oral fluid samples from 127 hepatitis C virus (HCV)-positive and 31 HCV-negative patients were tested for the presence of anti-HCV using the Ortho HCV 3.0 ELISA. Using the immunoglobulin G (IgG)-specific detection antibody provided with the HCV 3.0 ELISA we attained 100% sensitivity and specificity with serum samples; however, sensitivity in oral fluid samples was only 81%. By modifying the HCV 3.0 ELISA to utilize a secondary antibody cocktail that recognizes not only IgG but IgA and IgM as well, we attained 100% specificity and sensitivity with oral fluid samples.
Increased Levels of Inflammatory Mediators in Children and Adults Infected withVibrio choleraeO1 and O139
Authors: Qadri Firdausi , Raqib Rubhana , Ahmed Firoz , Rahman Taufiqur , Wenneras Christine , Kumar Das Swadesh , Alam Nur Haque , Mathan Minnie M. , Svennerholm Ann-Mari
Published: July 2002

Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected withVibrio choleraeO1 andV. choleraeO139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E2(PGE2), leukotriene B4(LTB4), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO·) metabolites (nitrite and nitrate [NO2−and NO3−]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F2αremained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected withV. choleraeO1 or the encapsulatedV. choleraeO139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE2, LTB4, and NO·, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected withV. choleraeO1 and O139.
Sensitive and Specific Identification ofNeospora caninumInfection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29
Authors: Howe Daniel K. , Tang Keliang , Conrad Patricia A. , Sverlow Karen , Dubey J. P. , Sibley L. David
Published: July 2002

Neosporosis is an economically important disease of dairy cattle caused by the protozoanNeospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar betweenN. caninumand closely related parasitesToxoplasma gondiiandSarcocystis cruzi.To provide a sensitive and specific assay for detecting antibodies toN. caninumin the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to theSAG1gene previously characterized fromT. gondii.An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected withN. caninum(optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected withT. gondiiorS. cruziwere negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.
Infection of Tick Cells and Bovine Erythrocytes with One Genotype of the Intracellular EhrlichiaAnaplasma marginaleExcludes Infection with Other Genotypes
Authors: de la Fuente José , Garcia-Garcia Jose C. , Blouin Edmour F. , Saliki Jeremiah T. , Kocan Katherine M.
Published: July 2002

Anaplasma marginale,a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the United States. Many geographic isolates ofA. marginalethat occur in the United States are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. Recent studies (G. H. Palmer, F. R. Rurangirwa, and T. F. McElwain, J. Clin. Microbiol. 39:631-635, 2001) of anA. marginale-infected herd of cattle in an area of endemicity demonstrated that multiplemsp1αgenotypes were present but that only one genotype was found per individual bovine. These findings suggested that infection of cattle with other genotypes was excluded. The present study was undertaken to confirm the phenomenon of infection exclusion ofA. marginalegenotypes in infected bovine erythrocytes and cultured tick cells. Two tick-transmissible isolates ofA. marginale, one from Virginia and one from Oklahoma, were used for these studies. In two separate trials, cattle inoculated with equal doses of the two isolates developed infection with only one genotype. Tick cell cultures inoculated with equal doses of the two isolates became infected with only the Virginia isolate ofA. marginale. When cultures were inoculated with different ratios of the Oklahoma and Virginia isolates ofA. marginale, the isolate inoculated in the higher ratio became established and excluded infection with the other. When cultures with established infections of one isolate were subsequently infected with the other, only the established isolate was detected. We documented infection exclusion during initial infection in cell culture by labeling each isolate with a different fluorescent dye. After 2 days in culture, only a single isolate was detected per cell by fluorescence microscopy. Finally, whenAnaplasma ovisinfections were established in cultures that were subsequently inoculated with the Virginia or Oklahoma isolate ofA. marginale,A. marginaleinfection was excluded. These studies confirm that infection exclusion occurs withA. marginalein bovine erythrocytes and tick cells, resulting in the establishment of only one genotype, and appears to be the first report of infection exclusion forAnaplasmaandEhrlichiaspecies.