bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacteria Associated with Hazelnut (
Corylus avellana
L.) Decline Are of Two Groups:
Pseudomonas avellanae
and Strains Resembling
P. syringae
pv. syringae
Authors: Scortichini Marco , Marchesi Ugo , Rossi Maria Pia , Di Prospero Paola
Published: February 2003

A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the
syrB
gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with
Hin
fI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the
syrB
gene, are pathogenically restricted to
Corylus avellana
, and belong to
Pseudomonas avellanae
. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to
Pseudomonas syringae
pv. syringae.
Molecular Analysis of
Dehalococcoides
16S Ribosomal DNA from Chloroethene-Contaminated Sites throughout North America and Europe
Authors: Hendrickson Edwin R. , Payne Jo Ann , Young Roslyn M. , Starr Mark G. , Perry Michael P. , Fahnestock Stephen , Ellis David E. , Ebersole Richard C.
Published: February 2003

The environmental distribution of
Dehalococcoides
group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the
Dehalococcoides
group by using a PCR assay developed to detect
Dehalococcoides
16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the
Dehalococcoides
group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites.
Dehalococcoides
sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-
cis
-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that
Dehalococcoides
sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the
Dehalococcoides
16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the
Dehalococcoides
group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.
Numerical Dominance and Phylotype Diversity of Marine
Rhodobacter
Species during Early Colonization of Submerged Surfaces in Coastal Marine Waters as Determined by 16S Ribosomal DNA Sequence Analysis and Fluorescence In Situ Hybridization
Authors: Dang Hongyue , Lovell Charles R.
Published: February 2003

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine
Rhodobacter
group of the α subdivision of the division
Proteobacteria
(α-
Proteobacteria
). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the
Bacteria
domain probe EUB338.
Archaea
were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the
Proteobacteria
examined, the α-
Proteobacteria
were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine
Rhodobacter
group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine
Rhodobacter
group organisms. These results demonstrated the quantitative importance of the marine
Rhodobacter
group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.
Phylogenetic Composition of Bacterioplankton Assemblages from the Arctic Ocean
Authors: Bano Nasreen , Hollibaugh James T.
Published: February 2003

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domainBacteria: α (36%)-, γ (32%)-, δ (14%)-, and ε (1%)-Proteobacteria;Cytophaga-Flexibacter-Bacteroidesspp. (9%);Verrucomicrobiumspp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 andRoseobactergroups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related toCytophagaandPolaribacterspecies were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) withAlteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) withOceanospirillumsp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to α-Proteobacteria(dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to γ-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.
Displacement of
Escherichia coli
O157:H7 from Rumen Medium Containing Prebiotic Sugars
Authors: de Vaux Albane , Morrison Mark , Hutkins Robert W.
Published: February 2003

A fed-batch, anaerobic culture system was developed to assess the behavior of
Escherichia coli
O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10
10
and 10
4
cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of
E. coli
O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10
6
to 10
9
CFU/ml within 24 h. Most strains of
E. coli
O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude
E. coli
O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter,
E. coli
O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as
l
-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever
E. coli
O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of
E. coli
O157:H7 by the addition of “nonfermentable” sugars and that this approach may be effective as a preharvest strategy for reducing carriage of
E. coli
O157:H7 in the rumen.
Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of
Proteus mirabilis
and
Escherichia coli
Authors: Hoischen Christian , Fritsche Christine , Gumpert Johannes , Westermann Martin , Gura Katleen , Fahnert Beatrix
Published: February 2003

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of
Escherichia coli
and
Proteus mirabilis
. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from
E. coli
(lactose permease LacY, preprotein translocase SecY) and
P. mirabilis
(curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 μg ml
−1
, with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.
Osmoregulation and Fungicide Resistance: the
Neurospora crassa os-2
Gene Encodes a
HOG1
Mitogen-Activated Protein Kinase Homologue
Authors: Zhang Yan , Lamm Randy , Pillonel Christian , Lam Stephen , Xu Jin-Rong
Published: February 2003

Neurospora crassa
osmosensitive (
os
) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that
os-2
and
os
-
5
mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the
os-2
gene by using sib selection.
os-2
encodes a putative mitogen-activated protein (MAP) kinase homologous to
HOG1
and can complement the osmosensitive phenotype of a
Saccharomyces cerevisiae hog1
mutant. We sequenced three
os-2
alleles and found that all of them were null with either frameshift or nonsense point mutations. An
os-2
gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely,
os-2
mutants transformed with the wild-type
os-2
gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in
os-2
mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of
N. crassa
is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive
os-2
MAP kinase pathway.
Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis
Authors: Bühler Bruno , Witholt Bernard , Hauer Bernhard , Schmid Andreas
Published: February 2003

Xylene monooxygenase of
Pseudomonas putida
mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant
Escherichia coli
expressing the monooxygenase genes
xylM
and
xylA
catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using
E. coli
expressing
xylMA
, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of
E. coli
was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.
Sources and Species of
Cryptosporidium
Oocysts in the Wachusett Reservoir Watershed
Authors: Jellison Kristen L. , Hemond Harold F. , Schauer David B.
Published: February 2003

Understanding the behavior of
Cryptosporidium
oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen
Cryptosporidium
parvum
from other
Cryptosporidium
species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.
l-Glucitol Catabolism inStenotrophomonas maltophiliaAc
Authors: Brechtel Elke , Huwig Alexander , Giffhorn Friedrich
Published: February 2003

The carbohydrate catabolism of the bacteriumStenotrophomonas maltophiliaAc (previously namedPseudomonassp. strain Ac), which is known to convert the unnatural polyoll-glucitol tod-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channelsl-glucitol viad-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified.d-Sorbose was converted by C-3 epimerization tod-tagatose, which, in turn, was isomerized tod-galactose.d-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation ofd-galactose tod-galactonate, its dehydration to 2-keto-3-deoxy-d-galactonate, and its phosphorylation to 2-keto-3-deoxy-d-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate andd-glycerate 3-phosphate as the central metabolic intermediates.
Expression of ActA, Ami, InlB, and Listeriolysin O in
Listeria monocytogenes
of Human and Food Origin
Authors: Jacquet C. , Gouin E. , Jeannel D. , Cossart P. , Rocourt J.
Published: February 2003

Expression of proteins involved in the adhesion of
Listeria monocytogenes
to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of
L. monocytogenes
strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90;
P
= 1 × 10
−4
). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89;
P
= 1 × 10
−2
) and central nervous system infections were associated with group ActA2 strains (OR = 3.04;
P
= 1 × 10
−3
) and group ActA3 strains (OR = 3.91;
P
= 1 × 10
−3
).
Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance
Authors: Di Cagno Raffaella , De Angelis Maria , Lavermicocca Paola , De Vincenzi Massimo , Giovannini Claudio , Faccia Michele , Gobbetti Marco
Published: February 2003

Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns,
Lactobacillus alimentarius
15M,
Lactobacillus brevis
14G,
Lactobacillus sanfranciscensis
7A, and
Lactobacillus hilgardii
51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by
L. alimentarius
15M,
L. brevis
14G, and
L. sanfranciscensis
7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of
L. alimentarius
15M and
L. brevis
14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.
Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from
Comamonas
sp. Strain JS765
Authors: Lessner Daniel J. , Johnson Glenn R. , Parales Rebecca E. , Spain Jim C. , Gibson David T.
Published: February 2003

Comamonas
sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase
NBZ
, Ferredoxin
NBZ
, Oxygenase
NBZα
, and Oxygenase
NBZβ
, with the gene designations
nbzAa
,
nbzAb
,
nbzAc
, and
nbzAd
, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from
Pseudomonas
sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase
NBZ
revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.
Tetrachloroethene Dehalorespiration and Growth of
Desulfitobacterium frappieri
TCE1 in Strict Dependence on the Activity of
Desulfovibrio fructosivorans
Authors: Drzyzga Oliver , Gottschal Jan C.
Published: February 2003

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium
Desulfovibrio fructosivorans
and the dehalorespiring
Desulfitobacterium frappieri
TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to
cis
-dichloroethene (
cis
-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to
cis
-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H
2
measurements of the gas phase and by the production of methane after the addition of a third organism,
Methanobacterium formicicum
.
Identification and Characterization of Pathogenic
Aeromonas veronii
Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh
Authors: Rahman Mokhlasur , Colque-Navarro Patricia , Kühn Inger , Huys Geert , Swings Jean , Möllby Roland
Published: February 2003

Sparse information is available on the virulence factors of
Aeromonas
strains isolated from diseased fish, from the environment, and from humans. In the present study, 52
Aeromonas
isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be
Aeromonas veronii
biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi
Aeromonas
isolates. The 52
Aeromonas
isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of
A. veronii
biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.
Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea
Authors: Pernthaler Annelie , Preston Christina M. , Pernthaler Jakob , DeLong Edward F. , Amann Rudolf
Published: February 2003

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (
Euryarchaeota
) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.
Characterization of the
norB
Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium
Synechocystis
sp. Strain PCC6803
Authors: Büsch Andrea , Friedrich Bärbel , Cramm Rainer
Published: February 2003

A
norB
gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium
Synechocystis
sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier
Ralstonia
eutropha
. Heterologous complementation of a nitric oxide reductase-negative mutant of
R
.
eutropha
with
norB
from
Synechocystis
restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of
Synechocystis
wild-type cells. Virtually no nitric oxide reductase activity was present in a
norB
-negative mutant of
Synechocystis
. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for
Synechocystis
when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor
norB
expression in
Synechocystis
. Transcription of
norB
was not enhanced by the addition of the NO-generating agent sodium nitroprusside.
Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu
Authors: Lamberg Arja , Nieminen Sari , Qiao Mingqiang , Savilahti Harri
Published: February 2003

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10
4
to 10
6
CFU/μg of input transposon DNA in the four species tested, i.e.,
Escherichia coli
,
Salmonella enterica
serovar Typhimurium,
Erwinia carotovora
, and
Yersinia enterocolitica
. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.
Impact of Transport Crate Reuse and of Catching and Processing on
Campylobacter
and
Salmonella
Contamination of Broiler Chickens
Authors: Slader J. , Domingue G. , Jørgensen F. , McAlpine K. , Owen R. J. , Bolton F. J. , Humphrey T. J.
Published: February 2003

The influence of transport, catching, and processing on contamination of broiler chickens with
Salmonella
and
Campylobacter
was investigated. Transport crates were reused with high frequency and were often still contaminated with
Salmonella
and
Campylobacter
when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously
Campylobacter
- and
Salmonella
-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The
Campylobacter
types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with
Campylobacter
(
P
< 0.001).
Identification, Characterization, and Expression of a Second, Bicistronic, Operon Involved in the Production of Lactocin S in
Lactobacillus sakei
L45
Authors: Skaugen Morten , Andersen Elizabeth L. , Christie Vigdis H. , Nes Ingolf F.
Published: February 2003

Through the analysis of spontaneous insertion mutants of
Lactobacillus sakei
L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed
lasXY
, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (
lasA-W
) and is transcribed in the opposite direction. The proximal of the two newly identified genes,
lasX
, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the
lasX
mutants have a strongly reduced level of
lasA
and
lasA-W
mRNA, thus indicating the likely cause of the Bac
−
phenotype of these mutants. The second ORF in the operon,
lasY
, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the
lasXY
operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the −35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of
lasA
and downstream genes.
Frequency of Bacteriocin Resistance Development and Associated Fitness Costs in
Listeria monocytogenes
Authors: Gravesen A. , Jydegaard Axelsen A.-M. , Mendes da Silva J. , Hansen T. B. , Knøchel S.
Published: February 2003

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in
Listeria monocytogenes
to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10
−6
. However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10
−7
to 10
−2
. Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10°C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 × 10
−8
). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5°C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5°C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.
Genomic Diversity within the Genus
Pediococcus
as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis
Authors: Simpson P. J. , Stanton C. , Fitzgerald G. F. , Ross R. P.
Published: February 2003

The genomic diversity of 33 previously assigned strains from six species within the genus
Pediococcus
was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes
Apa
I,
Not
I, and
Asc
I produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the
Not
I and
Asc
I macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with
Asc
I, all
Pediococcus parvulus
strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three
P. damnosus
and two
P. pentosaceus
strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.
Use of a Marker Organism To Model the Spread of Central Nervous System Tissue in Cattle and the Abattoir Environment during Commercial Stunning and Carcass Dressing
Authors: Daly D. J. , Prendergast D. M. , Sheridan J. J. , Blair I. S. , McDowell D. A.
Published: February 2003

Due to concerns about a link between variant Creutzfeldt-Jakob disease in humans and similar prion protein-induced disease in cattle, i.e., bovine spongiform encephalopathy (BSE), strict controls are in place to exclude BSE-positive animals and/or specified risk materials including bovine central nervous system (CNS) tissue from the human food chain. However, current slaughter practice, using captive bolt guns, may induce disruption of brain tissues and mobilize CNS tissues into the bovine circulatory system, leading to the dispersion of CNS tissues (including prion proteins) throughout the derived carcass. This project used a marker (antibiotic-resistant) strain of
Pseudomonas fluorescens
to model the effects of commercial captive bolt stunning procedures on the movement of mobilized CNS material within slaughtered animals and the abattoir environment. The marker organism, introduced by injection through the bolt entry aperture or directly using a cartridge-fired captive bolt, was detected in the slaughter environment immediately after stunning and in the abattoir environment at each subsequent stage of the slaughter-dressing process. The marker organism was also detected on the hands of operatives; on slaughter equipment; and in samples of blood, organs, and musculature of inoculated animals. There were no significant differences between the results obtained by the two inoculation methods (
P
< 0.05). This study demonstrates that material present in, or introduced into, the CNS of cattle during commercial captive bolt stunning may become widely dispersed across the many animate and inanimate elements of the slaughter-dressing environment and within derived carcasses including meat entering the human food chain.
Optimization of Reverse Transcriptase PCR To Detect Viable Shiga-Toxin-Producing
Escherichia coli
Authors: McIngvale S. C. , Elhanafi D. , Drake M. A.
Published: February 2003

The ability of reverse transcriptase PCR (RT-PCR) to detect viable Shiga-toxin-producing
Escherichia coli
(STEC) was investigated. Four primer sets, each targeting a specific region in the
slt-II
operon, were evaluated for their stringency and specificity for
slt-II
mRNA. STEC were evaluated for toxin expression under various conditions, including cell growth phase, growth medium, incubation temperature, and aeration. Following primer optimization, STEC were inoculated into Trypticase soy broth and cooked ground beef enrichments. Cells were harvested and RNA or DNA was extracted at 4, 8, 12, and 24 h. RT-PCR or PCR was conducted, and the products were visualized by gel electrophoresis and by Southern blots. mRNA targets were detected in 12-h cooked ground meat enrichments with an initial inoculum of 1 CFU/g. These results indicate that RT-PCR of
E. coli slt-II
mRNA is useful for detection of viable STEC in ground beef.
Identification of a Bacterium That Specifically Catalyzes the Reductive Dechlorination of Polychlorinated Biphenyls with Doubly Flanked Chlorines
Authors: Wu Qingzhong , Watts Joy E. M. , Sowers Kevin R. , May Harold D.
Published: February 2003

A microorganism whose growth is linked to the dechlorination of polychlorinated biphenyls (PCBs) with doubly flanked chlorines was identified. Identification was made by reductive analysis of community 16S ribosomal DNA (rDNA) sequences from a culture enriched in the presence of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB), which was dechlorinated at the
para
position. Denaturing gradient gel electrophoresis (DGGE) analysis of total 16S rDNA extracted from the culture led to identification of three operational taxonomic units (OTUs 1, 2, and 3). OTU 1 was always detected when 2,3,4,5-CB or other congeners with doubly flanked chlorines were present and dechlorinated. Only OTUs 2 and 3 were detected in the absence of PCBs and when other PCBs (i.e., PCBs lacking doubly flanked chlorines) were not dechlorinated. Partial sequences of OTUs 2 and 3 exhibited 98.2% similarity to the sequence of “
Desulfovibrio caledoniensis
” (accession no.
DCU53465
). A sulfate-reducing vibrio isolated from the culture generated OTUs 2 and 3. This organism could not dechlorinate 2,3,4,5-CB. From these results we concluded that OTU 1 represents the dechlorinating bacterium growing in a coculture with a
Desulfovibrio
sp. The 16S rDNA sequence of OTU 1 is most similar to the 16S rDNA sequence of bacterium
o
-17 (89% similarity), an
ortho
-PCB-dechlorinating bacterium. The PCB dechlorinator, designated bacterium DF-1, reductively dechlorinates congeners with doubly flanked chlorines when it is supplied with formate or H
2
-CO
2
(80:20).
Characterization of Glycine Betaine Porter I from
Listeria monocytogenes
and Its Roles in Salt and Chill Tolerance
Authors: Mendum Mary Lou , Smith Linda Tombras
Published: February 2003

Listeria monocytogenes
is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in
L. monocytogenes
is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na
+
-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed
L. monocytogenes
grown in the presence of glycine betaine.
Biodiversity of the Bacterial Flora on the Surface of a Smear Cheese
Authors: Brennan Noelle M. , Ward Alan C. , Beresford Thomas P. , Fox Patrick F. , Goodfellow Michael , Cogan Timothy M.
Published: February 2003

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with
Brevibacterium linens
BL2) and noninoculated (not deliberately inoculated with
B. linens
BL2) cheese were investigated. The results show that, contrary to accepted belief,
B. linens
is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative
Staphylococcus
spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of
Corynebacterium casei
(50.2% of isolates),
Corynebacterium mooreparkense
(26% of isolates), and
Microbacterium gubbeenense
(12.8% of isolates). In addition, five of the isolates from the inoculated cheese were
Corynebacterium flavescens
. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species.
C. mooreparkense
and
C. casei
grew at pH values below 4.9 in the presence of 8% NaCl, while
M. gubbeenense
did not grow below pH 5.8 in the presence of 5 to 10% NaCl.
B. linens
BL2 was not recovered from the inoculated cheese because it was inhibited by all the
Staphylococcus
isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.