bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of Biosurfactant-Producing Bacteria in Undisturbed and Contaminated Arid Southwestern Soils
Authors: Bodour Adria A. , Drees Kevin P. , Maier Raina M.
Published: June 2003

Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R
2
A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a
Flavobacterium
sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.
Assessment of Bioavailability of Soil-Sorbed Atrazine
Authors: Park Jeong-Hun , Feng Yucheng , Ji Pingsheng , Voice Thomas C. , Boyd Stephen A.
Published: June 2003

Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact. Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine. Three atrazine-degrading bacteria that utilized atrazine as a sole N source (
Pseudomonas
sp. strain ADP,
Agrobacterium radiobacter
strain J14a, and
Ralstonia
sp. strain M91-3) were used in the bioavailability assays. Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO
2
production over time. Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients. Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite. The desorption profiles were well described by the three-site desorption model. In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine. However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed. Even the assumption of instantaneous desorption could not account for the elevated rates. A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations. Characteristics of high sorbed-phase concentration, chemotaxis, and attachment of cells to soil particles seem to contribute to the bioavailability of soil-sorbed atrazine.
Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the
Lactobacillus casei
Group
Authors: Provencher Cathy , LaPointe Gisèle , Sirois Stéphane , Van Calsteren Marie-Rose , Roy Denis
Published: June 2003

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the
Lactobacillus casei
group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of
Lactobacillus rhamnosus
,
L. casei
,
Lactobacillus zeae
, and
Streptococcus thermophilus
. The priming GT gene was detected in the genome of both non-EPS-producing (EPS
−
) and EPS-producing (EPS
+
) strains of
L. rhamnosus
. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS
+
bacteria. Specific primers designed from the
L. rhamnosus
RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS
+
strains of
L. rhamnosus
. Phylogenetic analysis revealed that
Lactobacillus
spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of
L. rhamnosus
with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.
Characterization of Urease-Positive Thermophilic
Campylobacter
Subspecies by Multilocus Enzyme Electrophoresis Typing
Authors: Matsuda Motoo , Kaneko Aki , Stanley Timothy , Millar B. Cherie , Miyajima M. , Murphy Philip G. , Moore John E.
Published: June 2003

Thirty-one urease-positive thermophilic
Campylobacter
(UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three
Campylobacter lari
isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and
l
-phenylalanyl-
l
-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three
C. lari
isolates examined, and no ETs were shared by UPTC and
C. lari
isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the
C. lari
cluster.
Microbiological Evaluation of a Range of Disinfectant Products To Control Mixed-Species Biofilm Contamination in a Laboratory Model of a Dental Unit Water System
Authors: Walker J. T. , Bradshaw D. J. , Fulford M. R. , Marsh P. D.
Published: June 2003

Dental unit water system (DUWS) tubing harbors complex multispecies biofilms that are responsible for high microbial levels at the distal outlet. The aim of this study was to use an established biofilm laboratory model to simulate biofouling of DUWS to evaluate practical, cost-effective, and evidence-based methods of microbial decontamination. Reproducible biofilms were developed in the model over 14 days; decontamination was assessed using total viable counts (TVC) and microscopic-image analysis techniques to view the inner surface of tubing. Flushing did not reduce the biofilm coverage or TVC. Combizyme and ozone did not completely eliminate the viable bacteria (70 and 65% reduction in biofilm TVC, respectively), nor did they remove the biofilm (45 and 57% reduction in biofilm coverage, respectively). Chlorhexidine and Bio2000 (active agent: ethanol and chlorhexidine), Tegodor and Gigasept Rapid (aldehyde based), and Grotanol (hydroxide based) completely eliminated the TVC but did not completely remove biofilm (31, 53 33, 34, and 64.9% reduction of biofilm coverage, respectively). Other products including Grotanol Flussig (phenol based), Betadine (povidone-iodine based), Alpron (chlorite based), and the hydroxide-containing products Sporklenz, Sterilex Ultra, Dialox, Sterilox, Sanosil, Oxigenal, and Grotanat Bohrerbad resulted in a 100% reduction in the biofilm TVC and a >95% reduction in biofilm coverage. The study demonstrated that while many disinfectants achieve a sufficient reduction in TVC they may not necessarily remove unwanted biofilm from the tubing surfaces as tested in this laboratory-controlled biofilm model.
Interactions of Insecticidal Toxin Gene Products from
Xenorhabdus nematophilus
PMFI296
Authors: Sergeant Martin , Jarrett Paul , Ousley Margaret , Morgan J. Alun W.
Published: June 2003

Four genes on a genomic fragment from
Xenorhabdus nematophilus
PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in
Escherichia coli
, and the insecticidal activity of the lysates was determined. The combined four genes (
xptA1
,
xptA2
,
xptB1
, and
xptC1
), in
E. coli
, showed activity towards
Pieris brassicae
,
Pieris rapae
, and
Heliothis virescens
. The genes
xptA1
,
xptB1
, and
xptC1
were involved in expressing activity towards
P. rapae
and
P. brassicae
, while the genes
xptA2
,
xptB1
, and
xptC1
were needed for activity towards
H. virescens
. When each of these three genes was expressed individually in
E. coli
and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes
xptB1
and
xptC1
were expressed in the same
E. coli
cell and this cell lysate was mixed with cells expressing
xptA1
, activity was restored to
P. rapae
and
P. brassicae
. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards
H. virescens.
Individual gene disruptions in
X. nematophilus
PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either
xptA1
or
xptA2
for their respective disruptions or
E. coli
expressing both
xptB1
and
xptC1
for individual disruptions of either of these genes. The genes
xptA2
,
xptC1
, and
xptB1
were expressed as an operon in PMFI296 and inactivation of
xptA2
or
xptC1
resulted in silencing of downstream gene(s), while
xptA1
was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.
Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR
Authors: Baldwin Brett R. , Nakatsu Cindy H. , Nies Loring
Published: June 2003

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 10
2
copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.
Development of a
Listeria monocytogenes
EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates
Authors: Ramnath Manilduth , Rechinger K. Björn , Jänsch Lothar , Hastings John W. , Knøchel Susanne , Gravesen Anne
Published: June 2003

A partially annotated proteome reference map of the food pathogen
Listeria monocytogenes
was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the
L. monocytogenes
EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in
L. monocytogenes
EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the
L. monocytogenes
EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.
Determination of the Domain of the
Lactobacillus delbrueckii
subsp.
bulgaricus
Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the
prtB
Gene in
Lactococcus lactis
Authors: Germond Jacques-Edouard , Delley Michèle , Gilbert Christophe , Atlan Danièle
Published: June 2003

Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of
Lactobacillus delbrueckii
subsp.
bulgaricus
differs from other CSPs synthesized by lactic acid bacteria. Expression of the
prtB
gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP
−
PrtM
−
) of
Lactococcus lactis
subsp.
cremoris
. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables
L. lactis
MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.
Increasing the Oxidative Stress Response AllowsEscherichia coliTo Overcome Inhibitory Effects of Condensed Tannins
Authors: Smith Alexandra H. , Imlay James A. , Mackie Roderick I.
Published: June 2003

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towardsEscherichia coliwas determined by using an extract fromAcacia mearnsii(Black wattle) as a source of condensed tannins (proanthocyanidins).E. coligrowth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium.E. colimutants that lacked HPI, the major catalase, were especially sensitive to tannins, whileoxyRmutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic toE. coliin aerobic medium primarily because they generate H2O2. The oxidative stress response helpsE. colistrains to overcome their inhibitory effect.
Microbial Diversity of Biofilms in Dental Unit Water Systems
Authors: Singh Ruby , Stine O. Colin , Smith David L. , Spitznagel John K. , Labib Mohamed E. , Williams Henry N.
Published: June 2003

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the β and γ, but not the α, subclasses of
Proteobacteria.
In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera
Afipia
(28%) and
Sphingomonas
(16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included
Leptospira
(20%),
Sphingomonas
(14%),
Bacillus
(7%),
Escherichia
(6%),
Geobacter
(5%), and
Pseudomonas
(5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.
Metabolic Engineering of
Escherichia coli
for Production of Enantiomerically Pure (
R
)-(−)-Hydroxycarboxylic Acids
Authors: Lee Sang Yup , Lee Young
Published: June 2003

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in
Escherichia coli
by introducing the
Ralstonia eutropha
PHA biosynthesis operon along with the
R. eutropha
intracellular PHA depolymerase gene. By with this metabolically engineered
E. coli
, enantiomerically pure (
R
)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of
E. coli
and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (
R
)-hydroxycarboxylic acids from renewable resources.
1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in
Torula corallina
, and DHN-Melanin Inhibits Erythrose Reductase
Authors: Lee Jung-Kul , Jung Hyung-Moo , Kim Sang-Yong
Published: June 2003

The yeast
Torula corallina
is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the
T. corallina
cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of
T. corallina
is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from
T. corallina
was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in
T. corallina
.
Use of PCR for Direct Detection of
Campylobacter
Species in Bovine Feces
Authors: Inglis G. Douglas , Kalischuk Lisa D.
Published: June 2003

This study reports on the use of PCR to directly detect and distinguish
Campylobacter
species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR,
Campylobacter coli
,
C. fetus
,
C. hyointestinalis
, and
C. jejuni
were detected in all fecal samples inoculated at ≈10
4
CFU g
−1
, and 50 to 83% of the samples inoculated at ≈10
3
CFU g
−1
were positive. At ≈10
2
CFU g
−1
,
C. fetus
,
C. hyointestinalis
, and
C. jejuni
(17 to 50% of the samples) but not
C. coli
were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of
Campylobacter
were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were
C. jejuni
(152 isolates) and
C. lanienae
(42 isolates), but isolates of
C. fetus
subsp.
fetus
,
Arcobacter butzleri
, and
A. skirrowii
also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers,
Campylobacter
DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers,
C. jejuni
and
C. lanienae
were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either
C. coli
,
C. fetus
, or
C. hyointestinalis
detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting
C. lanienae
(17%) but not
C. jejuni
. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of
Campylobacter
species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.
Oxygen-Insensitive Nitroreductases NfsA and NfsB ofEscherichia coliFunction under Anaerobic Conditions as Lawsone-Dependent Azo Reductases
Authors: Rau Jörg , Stolz Andreas
Published: June 2003

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells ofEscherichia coliresulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone byE. coliand thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase ofE. coli(NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.
Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect
Salmonella enterica
Serotype Enteritidis in Poultry
Authors: De Medici Dario , Croci Luciana , Delibato Elisabetta , Di Pasquale Simona , Filetici Emma , Toti Laura
Published: June 2003

The objective of this study was to develop a rapid, reproducible, and robust method for detecting
Salmonella enterica
serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (

T
m

) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the

T
m

, which was consistently specific for the amplicon obtained; the mean peak

T
m

obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10
3
to 10
8
CFU/ml) showed good linearity (
R
2
= 0.9767) and a sensitivity limit of less than 10
3
CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.
Secondary Metabolites of
Flustra foliace
a and Their Influence on Bacteria
Authors: Peters Lars , König Gabriele M. , Wright Anthony D. , Pukall Rüdiger , Stackebrandt Erko , Eberl Leo , Riedel Kathrin
Published: June 2003

The North Sea bryozoan
Flustra foliacea
was investigated to determine its secondary metabolite content. Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified. Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11). All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments. Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from
F. foliacea
but only weak activities against all of the terrestrial bacteria. By using the biosensors
Pseudomonas putida
(pKR-C12),
P. putida
(pAS-C8), and
Escherichia coli
(pSB403) the antagonistic effect on
N
-acyl-homoserine lactone-dependent quorum-sensing systems was investigated. Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 μg/ml.
Nitric Oxide Reductase (
norB
) Genes from Pure Cultures and Environmental Samples
Authors: Braker Gesche , Tiedje James M.
Published: June 2003

A PCR-based approach was developed to recover nitric oxide (NO) reductase (
norB
) genes as a functional marker gene for denitrifying bacteria.
norB
database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome
bc
-type complex (cNorB). The latter oxidizes cytochrome
c
, and the gene is localized adjacent to
norC
. While both
norB
types occur in denitrifying strains, the
qnorB
type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two
norB
types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the
norB
amplicons and failure to amplify
norB
from nondenitrifying strains. These primer sets also specifically amplified
norB
from freshwater and marine sediments. Pairwise comparison of amplified
norB
sequences indicated minimum levels of amino acid identity of 43.9% for
qnorB
and 38% for
cnorB
. Phylogenetic analysis confirmed the existence of two classes of
norB
genes, which clustered according to the respective primer set. Within the
qnorB
cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental
qnorB
clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms.
cnorB
clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.
Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato
Authors: Dianese Alexei C. , Ji Pingsheng , Wilson Mark
Published: June 2003

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogenPseudomonas syringaepv. tomato. This relationship was investigated further in this study by using the pathogenXanthomonas campestrispv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogenX. campestrispv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log10of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria andX. campestrispv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies withP. syringaepv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogenX. campestrispv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size ofX. campestrispv. vesicatoria is not related to disease severity and thatX. campestrispv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that ofP. syringaepv. tomato.
Use of Growth Indices from Radiometric Culture for Quantification of Sheep Strains ofMycobacterium aviumsubsp.paratuberculosis
Authors: Reddacliff Leslie A. , Nicholls Paul J. , Vadali Aparna , Whittington Richard J.
Published: June 2003

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains ofMycobacterium aviumsubsp.paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log10inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration ofM. aviumsubsp.paratuberculosisfrom field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of ±1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.
Ecology of
Vibrio vulnificus
in Estuarine Waters of Eastern North Carolina
Authors: Pfeffer Courtney S. , Hite M. Frances , Oliver James D.
Published: June 2003

While several studies on the ecology of
Vibrio vulnificus
in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of
V. vulnificus
in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of
Vibrio
spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured.
V. vulnificus
isolates, along with estuarine bacteria,
Vibrio
spp.,
Escherichia coli
organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including
V. vulnificus
. Isolates were confirmed as
V. vulnificus
by using hemolysin gene PCR or colony hybridization.
V. vulnificus
was isolated only when water temperatures were between 15 and 27°C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of
V. vulnificus
in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both
V. vulnificus
(47%) and
Vibrio
spp. (48%).
Use of Phospholipid Fatty Acids To Detect Previous Self-Heating Events in Stored Peat
Authors: Ranneklev Sissel Brit , Bååth Erland
Published: June 2003

The use of the phospholipid fatty acid (PLFA) composition of microorganisms to detect previous self-heating events was studied in naturally self-heated peat and in peat incubated under temperature-controlled conditions. An increased content of total PLFAs was found in self-heated peat compared to that in unheated peat. Two PLFAs, denoted T1 and T2, were detected only in the self-heated peat. Incubation of peat samples at 25 to 55°C for 4 days indicated that T1 and T2 were produced from microorganisms with different optimum temperatures. This was confirmed by isolation of bacteria at 55°C, which produced T2 but not T1. These bacteria produced another PLFA (denoted T3) which coeluted with 18:1ω7. T2 and T3 were identified as ω-cyclohexyltridecanoic acid and ω-cyclohexylundecanoic acid, respectively, indicating that the bacteria belonged to the genus
Alicyclobacillus
. T1 was tentatively identified as ω-cycloheptylundecanoic acid. T2 was detected 8 h after the peat incubation temperature was increased to 55°C, and maximum levels were found within 5 days of incubation. The PLFA 18:1ω7-T3 increased in proportion to T2. T1 was detected after 96 h at 55°C, and its level increased throughout the incubation period, so that it eventually became one of the dominant PLFAs after 80 days. In peat samples incubated at 55°C and then at 25°C, T1 and T2 disappeared slowly. After 3 months, detectable levels were still found. Incubation at 25°C after heating for 3 days at 55°C decreased the amounts of T2 and 18:1ω7-T3 faster than did incubation at 5°C. Thus, not only the duration and temperature during the heating event but also the storage temperature following heating are important for the detection of PLFAs indicating previous self-heating.
Bacterial Community Structure and Location in Stilton Cheese
Authors: Ercolini Danilo , Hill Philip J. , Dodd Christine E. R.
Published: June 2003

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of
Lactococcus lactis
,
Enterococcus faecalis
,
Lactobacillus plantarum
,
Lactobacillus curvatus
,
Leuconostoc mesenteroides
,
Staphylococcus equorum
, and
Staphylococcus
sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect
Lactococcus lactis
,
Lactobacillus plantarum
, and
Leuconostoc mesenteroides
in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core.
Lactobacillus plantarum
was detected only underneath the surface, while
Leuconostoc
microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.
Citrate Synthase Mutants of
Sinorhizobium fredii
USDA257 Form Ineffective Nodules with Aberrant Ultrastructure
Authors: Krishnan Hari B. , Kim Won-Seok , Sun-Hyung Jeong , Kim Kil Yong , Jiang Guoqiao
Published: June 2003

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the
gltA
gene that encodes citrate synthase in
Sinorhizobium fredii
USDA257, a symbiont of soybeans (
Glycine max
[L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including
Sinorhizobium meliloti
and
Rhizobium tropici
. Southern blot analysis revealed that the fast-growing
S. fredii
strains and
Rhizobium
sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome.
S. fredii
USDA257
gltA
mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257
gltA
gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of
S. fredii
USDA257 is essential for efficient soybean nodulation and nitrogen fixation.
Density-Dependent Sorting of Physiologically Different Cells of
Vibrio parahaemolyticus
Authors: Nishino Tomohiko , Nayak Binaya B. , Kogure Kazuhiro
Published: June 2003

A pure bacterial culture is composed of clonal cells in different physiological states. Separation of those subpopulations is critical for further characterization and for understanding various processes in the cultured cells. We used density-dependent cell sorting with Percoll to separate subpopulations from cultures of a marine bacterium,
Vibrio parahaemolyticus
. Cells from cultures in the exponential and stationary phases were fractionated according to their buoyant density, and their culturability and ability to maintain culturability under low-temperature and low-nutrient stress (stress resistance) were determined. The buoyant density of the major portion of the cells decreased with culture age. The culturability of stationary-phase cells increased with increasing buoyant density, but that of exponential-phase cells did not. Stress resistance decreased with increasing buoyant density regardless of the growth phase. The results indicate that density-dependent cell sorting is useful for separating subpopulations of different culturabilities and stress resistances. We expect that this method will be a powerful tool for analyzing cells in various physiological states, such as the viable but nonculturable state.
A C
35
Carotenoid Biosynthetic Pathway
Authors: Umeno Daisuke , Arnold Frances H.
Published: June 2003

Upon coexpression with
Erwinia
geranylgeranyldiphosphate (GGDP) synthase in
Escherichia coli
, C
30
carotenoid synthase CrtM from
Staphylococcus aureus
produces novel carotenoids with the asymmetrical C
35
backbone. The products of condensation of farnesyldiphosphate and GDP, C
35
structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C
40
or C
30
pathways accepted and converted the C
35
substrate, thus creating a C
35
carotenoid biosynthetic pathway in
E. coli
. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.