bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting
Salmonella
in Meat Products
Authors: Croci Luciana , Delibato Elisabetta , Volpe Giulia , De Medici Dario , Palleschi Giuseppe
Published: March 2004

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.
Characterization of a Thermostable
l
-Arabinose (
d
-Galactose) Isomerase from the Hyperthermophilic Eubacterium
Thermotoga maritima
Authors: Lee Dong-Woo , Jang Hyeung-Jin , Choe Eun-Ah , Kim Byoung-Chan , Lee Sang-Jae , Kim Seong-Bo , Hong Young-Ho , Pyun Yu-Ryang
Published: March 2004

The
araA
gene encoding
l
-arabinose isomerase (AI) from the hyperthermophilic bacterium
Thermotoga maritima
was cloned and overexpressed in
Escherichia coli
as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni
2+
affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent

K
m

values for
l
-arabinose and
d
-galactose were 31 and 60 mM, respectively; the apparent
V
max
values (at 90°C) were 41.3 U/mg (
l
-arabinose) and 8.9 U/mg (
d
-galactose), and the catalytic efficiencies (
k
cat
/

K
m

) of the enzyme were 74.8 mM
−1
· min
−1
(
l
-arabinose) and 8.5 mM
−1
· min
−1
(
d
-galactose). Although the
T. maritima
AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn
2+
and/or Co
2+
than in the absence of these ions. The enzyme carried out the isomerization of
d
-galactose to
d
-tagatose with a conversion yield of 56% for 6 h at 80°C.
Vanadium(V) Reduction by
Shewanella oneidensis
MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes
Authors: Myers Judith M. , Antholine William E. , Myers Charles R.
Published: March 2004

The metal-reducing bacterium
Shewanella oneidensis
MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.
PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the GenusGigaspora
Authors: de Souza Francisco A. , Kowalchuk George A. , Leeflang Paula , van Veen Johannes A. , Smit Eric
Published: March 2004

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genusGigasporato assess the use of this marker for the discrimination ofGigasporaisolates and ofGigasporaceaepopulations from environmental samples. Screening of 48Gigasporaisolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between differentGigasporaspecies. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates ofG. albida,G. gigantea, andG. margaritabut notG. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containingG. albida,G. candida,G. ramisporophora, andG. roseaand the other containingG. decipiensandG. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with aGigasporaceae-specific nested PCR approach, revealing a dominance ofG. margaritawithin this family.
Development of a Markerless Genetic Exchange Method for
Methanosarcina acetivorans
C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea
Authors: Pritchett Matthew A. , Zhang Jun Kai , Metcalf William W.
Published: March 2004

A new genetic technique for constructing mutants of
Methanosarcina acetivorans
C2A by using
hpt
as a counterselectable marker was developed. Mutants with lesions in the
hpt
gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the
hpt
gene into a Δ
hpt
host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the
M. acetivorans
chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Δ
proC
Δ
hpt
mutants, which were recovered at a frequency of 22%. Additionally, a
Methanosarcina-Escherichia
shuttle vector, encoding the
Escherichia coli proC
gene as a new selectable marker, was constructed for use in
proC
hosts. Finally, the markerless exchange method was used to recombine a series of
uidA
reporter gene fusions into the
M. acetivorans proC
locus. In vitro assay of β-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of
uidA
as a reporter gene in
Methanosarcina.
A >5,000-fold range of promoter activities could be measured by using
uidA
: the methyl-coenzyme M reductase operon fusion displayed ∼300-fold-higher activity than did the
serC
gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown
orf2
gene.
Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association
Authors: Kimbell Jennifer R. , McFall-Ngai Margaret J.
Published: March 2004

The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that
Vibrio fischeri
induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells.
Comparison of Prevalence and Antimicrobial Susceptibilities ofCampylobacterspp. Isolates from Organic and Conventional Dairy Herds in Wisconsin
Authors: Sato K. , Bartlett P. C. , Kaneene J. B. , Downes F. P.
Published: March 2004

The prevalence and antimicrobial susceptibilities ofCampylobacterspp.isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332Campylobacterisolates. The prevalence ofCampylobacterspp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms.Campylobacterprevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion ofCampylobacterisolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P= 0.042), but no difference was observed between organic and conventional farms. When we usedCampylobacterspp.as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.
Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination
Authors: Maluquer de Motes Carlos , Clemente-Casares Pilar , Hundesa Ayalkibet , Martín Margarita , Girones Rosina
Published: March 2004

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera
Mastadenovirus
and
Atadenovirus
and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.
Development and Testing of a DNA Macroarray To Assess Nitrogenase (
nifH
) Gene Diversity
Authors: Steward Grieg F. , Jenkins Bethany D. , Ward Bess B. , Zehr Jonathan P.
Published: March 2004

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (
nifH
) gene. Diverse
nifH
gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated
nifH
fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml
−1
, a half-saturation of signal at 0.26 ng ml
−1
, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (
r
= 0.98,
P
< 0.0001,
n
= 88). A mixed
nifH
target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (
r
= 0.941,
P
< 0.0001,
n
= 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.
Engineering
Lactococcus lactis
for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System
Authors: Gaspar Paula , Neves Ana Rute , Ramos Ana , Gasson Michael J. , Shearman Claire A. , Santos Helena
Published: March 2004

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of
Lactococcus lactis
was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS
Mtl
). Genes
mtlA
and
mtlF
were independently deleted by double-crossover recombination in strain
L. lactis
FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Δ
ldh
Δ
mtlA
and Δ
ldh
Δ
mtlF
) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo
13
C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS
Mtl
. Disruption of this system completely blocked mannitol transport in
L. lactis
, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.
Impact of Agricultural Practices on the
Zea mays
L. Endophytic Community
Authors: Seghers Dave , Wittebolle Lieven , Top Eva M. , Verstraete Willy , Siciliano Steven D.
Published: March 2004

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of
Zea mays
L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.
Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic
Mollicutes
Strains by Reverse Line Blot Hybridization
Authors: Wang Hui , Kong Fanrong , Jelfs Peter , James Gregory , Gilbert Gwendolyn L.
Published: March 2004

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (
Mycoplasma arginini
,
Mycoplasma fermentans
,
Mycoplasma hyorhinis
,
Mycoplasma orale
, and
Acholeplasma laidlawii
) and human infection (
Mycoplasma pneumoniae, Mycoplasma hominis
,
Mycoplasma genitalium
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage
Authors: Van Hamme Jonathan D. , Fedorak Phillip M. , Foght Julia M. , Gray Murray R. , Dettman Heather D.
Published: March 2004

The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C—S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C—S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound,
bis
-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C—S bonds. A unique bacterial strain,
Rhodococcus
sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C—S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including
Rhodococcus
sp. strain IGTS8, were all unable to cleave the C—S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C—S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.
Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time
Authors: Crump Byron C. , Hopkinson Charles S. , Sogin Mitchell L. , Hobbie John E.
Published: March 2004

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([
14
C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla
Proteobacteria
,
Bacteroidetes
, and
Actinobacteria
. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.
Optimization of Procedures for Counting Viruses by Flow Cytometry
Authors: Brussaard Corina P. D.
Published: March 2004

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10
−5
dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.
Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification
Authors: Völlenkle Christine , Weigert Stefan , Ilk Nicola , Egelseer Eva , Weber Viktoria , Loth Fritz , Falkenhagen Dieter , Sleytr Uwe B. , Sára Margit
Published: March 2004

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA fromBacillus sphaericusCCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed inEscherichia coliHMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.
Microbiological Characterization of Wet Wheat Distillers' Grain, with Focus on Isolation of Lactobacilli with Potential as Probiotics
Authors: Pedersen C. , Jonsson H. , Lindberg J. E. , Roos S.
Published: March 2004

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species
Lactobacillus amylolyticus
,
Lactobacillus panis
, and
Lactobacillus pontis
, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50°C, with
L. amylolyticus
being able to grow at temperatures as high as 54°C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of
L. panis
and
L. pontis
showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.
High Diversity among Environmental
Escherichia coli
Isolates from a Bovine Feedlot
Authors: Yang Hsiao-Hui , Vinopal Robert T. , Grasso Domenico , Smets Barth F.
Published: March 2004

Approximately 280
Escherichia coli
isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates. The
E. coli
subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints. A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the
E. coli
population richness at 128 OTUs. One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability. Motility, measured by a soft agar assay, had a very broad range among the
E. coli
population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient
r
= 0.619,
P
< 10
−4
) but not in Luria broth. Only an estimated 48% of the population possessed gene
agn43
, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium. We observed significantly more biofilm formation in both minimal medium and Luria broth for

agn43
+

strains, with a larger effect in minimal medium. This study represents an exhaustive inventory of extant
E. coli
population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.
2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant
Authors: Noel K. Dale , Box Jodie M. , Bonne Valerie J.
Published: March 2004

When
Rhizobium etli
CE3 was grown in the presence of
Phaseolus vulgaris
seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-
O
-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen.
R. etli
strain CE395 was isolated after Tn
5
mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-
O
-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with
P. vulgaris
. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-
O
-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than
lps
+
strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.
Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of
Escherichia coli
: Relationship with Cell Death
Authors: Mañas Pilar , Mackey Bernard M.
Published: March 2004

The relationship between a loss of viability and several morphological and physiological changes was examined with
Escherichia coli
strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.
Prevalence and Molecular Characterization of Tetracycline Resistance in
Enterococcus
Isolates from Food
Authors: Huys Geert , D'Haene Klaas , Collard Jean-Marc , Swings Jean
Published: March 2004

In the present study, a collection of 187
Enterococcus
food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species
Enterococcus faecalis
(
n
= 33),
E. durans
(
n
= 7),
E. faecium
(
n
= 3),
E. casseliflavus
(
n
= 1), and
E. gallinarum
(
n
= 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 μg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the
tet
(M) (
n
= 43),
tet
(L) (
n
= 16), and
tet
(S) (
n
= 1) genes. In 15 isolates, including all of those for which the MIC was 256 μg/ml, both
tet
(M) and
tet
(L) were found. Furthermore, all
tet
(M)-containing enterococci also harbored a member of the Tn
916
-Tn
1545
conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the
erm
(B) gene. Filter mating experiments revealed that 10
E. faecalis
isolates, 3
E. durans
isolates, and 1
E. faecium
isolate could transfer either
tet
(M),
tet
(L), or both of these genes to
E. faecalis
recipient strain JH2-2. In most cases in which only
tet
(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn
916
-Tn
1545
family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable
tet
(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of ≥99.6%. Two of these SHGs contained
tet
(M) mosaic structures previously found in Tn
916
elements and on
Lactobacillus
and
Neisseria
plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.
Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of
Clostridium thermocellum
Authors: Zhang Yi-Heng Percival , Lynd Lee R.
Published: March 2004

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from
Clostridium thermocellum
ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of
V
max
values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent
K

m

for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).
Characteristics of a New Enantioselective Thermostable Dipeptidase from
Brevibacillus borstelensis
BCS-1 and Its Application to Synthesis of a
d
-Amino-Acid-Containing Dipeptide
Authors: Baek Dae Heoun , Song Jae Jun , Kwon Seok-Joon , Park Chung , Jung Chang-Min , Sung Moon-Hee
Published: March 2004

A new thermostable dipeptidase gene was cloned from the thermophile
Brevibacillus borstelensis
BCS-1 by genetic complementation of the
d
-Glu auxotroph
Escherichia coli
WM335 on a plate containing
d
-Ala-
d
-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an
M
r
of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from
Listeria monocytogenes
. The enzyme was purified to homogeneity from recombinant
E. coli
WM335 harboring the dipeptidase gene from
B. borstelensis
BCS-1. Investigation of the enantioselectivity (
E
) to the P
1
and P
1
′ site of Ala-Ala revealed that the ratio of the specificity constant (
k
cat

/K
m

) for
l
-enantioselectivity to the P
1
site of Ala-Ala was 23.4 ± 2.2 [
E
= (
k
cat

/K
m

)

l,d

/(
k
cat

/K
m

)

d,d

], while the
d
-enantioselectivity to the P
1
′ site of Ala-Ala was 16.4 ± 0.5 [
E
= (
k
cat

/K
m

)

l,d

/(
k
cat

/K
m

)

l,l

] at 55°C. The enzyme was stable up to 55°C, and the optimal pH and temperature were 8.5 and 65°C, respectively. The enzyme was able to hydrolyze
l
-Asp-
d
-Ala,
l
-Asp-
d
-AlaOMe,
Z
-
d
-Ala-
d
-AlaOBzl, and
Z
-
l
-Asp-
d
-AlaOBzl, yet it could not hydrolyze
d
-Ala-
l
-Asp,
d
-Ala-
l
-Ala,
d
-AlaNH
2
, and
l
-AlaNH
2.
The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener
Z
-
l
-Asp-
d
-AlaOBzl.
Substantial Rise in the Prevalence of Lyme Borreliosis Spirochetes in a Region of Western Germany over a 10-Year Period
Authors: Kampen Helge , Rötzel Diana C. , Kurtenbach Klaus , Maier Walter A. , Seitz Hanns M.
Published: March 2004

More than a decade after a study on the transmission cycle of
Borrelia burgdorferi
sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult
Ixodes ricinus
ticks were collected again in three selected areas of the same low mountain range and examined for infection with
B. burgdorferi
sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for
B. burgdorferi
sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for
B. burgdorferi
sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of
B. burgdorferi
sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of
B. burgdorferi
sensu lato revealed high relative prevalences of
B. valaisiana
(identified in 43.1% of infected ticks) and
B. garinii
(32.3%), whereas
B. afzelii
(12.3%) and
B. burgdorferi
sensu stricto (1.5%) were relatively rare. We conclude that
B. burgdorferi
sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.
High-Throughput Metabolic Fingerprinting of Legume Silage Fermentations via Fourier Transform Infrared Spectroscopy and Chemometrics
Authors: Johnson Helen E. , Broadhurst David , Kell Douglas B. , Theodorou Michael K. , Merry Roger J. , Griffith Gareth W.
Published: March 2004

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm
−1
) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.
Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates
Authors: Matz Carsten , Deines Peter , Boenigk Jens , Arndt Hartmut , Eberl Leo , Kjelleberg Staffan , Jürgens Klaus
Published: March 2004

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of
Janthinobacterium lividum
and
Chromobacterium violaceum
produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic
Pseudomonas putida
MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of
C. violaceum
showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in
C. violaceum
is regulated by an
N
-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural
C. violaceum
AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.
Influence of Lipoteichoic Acid
d
-Alanylation on Protein Secretion in
Lactococcus lactis
as Revealed by Random Mutagenesis
Authors: Nouaille S. , Commissaire J. , Gratadoux J. J. , Ravn P. , Bolotin A. , Gruss A. , Le Loir Y. , Langella P.
Published: March 2004

Lactococcus lactis
, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in
L. lactis
. Random insertional mutagenesis was performed with
L. lactis
strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in
L. lactis
(∼40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the
dltA
gene (involved in
d
-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the
dltA
mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.