bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection, Quantitation, and Phylogenetic Analysis of Noroviruses in Japanese Oysters
Authors: Nishida Tomoko , Kimura Hirokazu , Saitoh Mika , Shinohara Michiyo , Kato Masahiko , Fukuda Shinji , Munemura Tetsuya , Mikami Toshiyuki , Kawamoto Ayumi , Akiyama Miho , Kato Yumiko , Nishi Kanako , Kozawa Kunihisa , Nishio Osamu
Published: October 2003

Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 10
2
copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.
Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate
Lactobacillus rhamnosus
BGT10
Authors: Pastar Irena , Tonic Ivana , Golic Natasa , Kojic Milan , van Kranenburg Richard , Kleerebezem Michiel , Topisirovic Ljubisa , Jovanovic Goran
Published: October 2003

A novel proteinase, PrtR, produced by the human vaginal isolate
Lactobacillus rhamnosus
strain BGT10 was identified and genetically characterized. The
prtR
gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its α
S1
- and β-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all
L. rhamnosus
strains tested. Proteinase extracts of the BGT10 strain obtained with Ca
2+
-free buffer at pH 6.5 were proteolytically active. The
prtR
promoter-like sequence was determined, and the minimal promoter region was defined by use of
prtR-gusA
operon fusions. The
prtR
expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.
Establishment of an Arbitrary PCR for Rapid Identification of Tn
917
Insertion Sites in
Staphylococcus epidermidis
: Characterization of Biofilm-Negative and Nonmucoid Mutants
Authors: Knobloch Johannes K.-M. , Nedelmann Max , Kiel Kathrin , Bartscht Katrin , Horstkotte Matthias A. , Dobinsky Sabine , Rohde Holger , Mack Dietrich
Published: October 2003

Transposon mutagenesis with the
Enterococcus faecalis
transposon Tn
917
is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn
917
insertion sites in
Staphylococcus epidermidis
with six independent, well-characterized biofilm-negative Tn
917
transposon mutants, which were clustered in the
icaADBC
gene locus or harbor Tn
917
in the regulatory gene
rsbU
. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn
917
insertion sites in the published
S. epidermidis
genomes. By using this technique, the Tn
917
insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn
917
is inserted into a gene homologous to the regulatory gene
purR
of
Bacillus subtilis
and
Staphylococcus aureus
. The Tn
917
insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of
B. subtilis
,
S. aureus
, and
Staphylococcus carnosus
, indicating an influence of the PTS on the mucoid phenotype in
S. epidermidis
.
Development of a PCR-Enzyme Immunoassay Oligoprobe Detection Method for
Toxoplasma gondii
Oocysts, Incorporating PCR Controls
Authors: Schwab Kellogg J. , McDevitt James J.
Published: October 2003

Infections caused by
Toxoplasma gondii
are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of
T. gondii
infection.
T. gondii
has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to
T. gondii
oocysts. Because of the lack of information regarding the prevalence of
T. gondii
in surface waters, there is a clear need for a rapid, sensitive method to detect
T. gondii
from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of
T. gondii
nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for
T. gondii
oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-
N
-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a
T. gondii
oocyst PCR detection method will provide a useful technique to estimate the levels of
T. gondii
oocysts present in surface waters.
Membrane Fluidity Adjustments in Ethanol-Stressed
Oenococcus oeni
Cells
Authors: Da Silveira M. Graça , Golovina Elena A. , Hoekstra Folkert A. , Rombouts Frank M. , Abee Tjakko
Published: October 2003

The effect of ethanol on the cytoplasmic membrane of
Oenococcus oeni
cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.
Substrate Preferences in Biodesulfurization of Diesel Range Fuels by
Rhodococcus
sp. Strain ECRD-1
Authors: Prince Roger C. , Grossman Matthew J.
Published: October 2003

The range of sulfur compounds in fuel oil and the substrate range and preference of the biocatalytic system determine the maximum extent to which sulfur can be removed by biodesulfurization. We show that the biodesulfurization apparatus in
Rhodococcus
sp. strain ECRD-1 is able to attack all isomers of dibenzothiophene including those with at least four pendant carbons, with a slight preference for those substituted in the α-position. With somewhat less avidity, this apparatus is also able to attack substituted benzothiophenes with between two and seven pendant carbons. Some compounds containing sulfidic sulfur are also susceptible to desulfurization, although we have not yet been able to determine their molecular identities.
Anaerobic Degradation of Flavonoids by
Clostridium orbiscindens
Authors: Schoefer Lilian , Mohan Ruchika , Schwiertz Andreas , Braune Annett , Blaut Michael
Published: October 2003

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as
Clostridium orbiscindens
by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated
C. orbiscindens
strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2′-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of
C. orbiscindens
in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of
C. orbiscindens
in 8 out of the 10 samples tested. The numbers ranged from 1.87 × 10
8
to 2.50 × 10
9
cells g of fecal dry mass
−1
, corresponding to a mean count of 4.40 × 10
8
cells g of dry feces
−1
.
Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from
Lactobacillus fermentum
BR11
Authors: Turner Mark S. , Hafner Louise M. , Walsh Terry , Giffard Philip M.
Published: October 2003

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from
Lactobacillus fermentum
BR11 has been identified by using an antiserum raised against whole
L. fermentum
BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from
Streptococcus agalactiae
, while the other protein, Mlp, is similar to the mucus binding protein Mub from
Lactobacillus reuteri
. It was shown that multiple copies of
mlp
exist in the genome of
L. fermentum
BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in
L. fermentum
. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His
6
). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The
mlp
promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His
6
and CFTR peptides or His
6
peptide, respectively. Therefore, these newly described proteins from
L. fermentum
BR11 have potential as protein production and targeting vectors.
Identification of
Naegleria fowleri
in Domestic Water Sources by Nested PCR
Authors: Marciano-Cabral Francine , MacLean Rebecca , Mensah Alex , LaPat-Polasko Laurie
Published: October 2003

The free-living amoeboflagellate
Naegleria fowleri
is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to
N. fowleri
and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for
N. fowleri
by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for
N. fowleri
by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for
N. fowleri
were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.
Mycotoxin Fumonisin B1Increases Intestinal Colonization by PathogenicEscherichia coliin Pigs
Authors: Oswald Isabelle P. , Desautels Clarisse , Laffitte Joëlle , Fournout Sylvie , Peres Sylvie Y. , Odin Marielle , Le Bars Pierrette , Le Bars Joseph , Fairbrother John M.
Published: October 2003

Fumonisin B1(FB1) is a mycotoxin that commonly occurs in maize. FB1causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans. In the present study, we examined the effect of dietary exposure to FB1on intestinal colonization by pathogenicEscherichia coliassociated with extraintestinal infection. Three-week-old weaned pigs were given FB1by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days. On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenicE. colistrain. All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination. Ingestion of FB1had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile. However, colonization of the small and large intestines by an extraintestinal pathogenicE. colistrain was significantly increased. Our results show that FB1is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food.
Stimulating the In Situ Activity of
Geobacter
Species To Remove Uranium from the Groundwater of a Uranium-Contaminated Aquifer
Authors: Anderson Robert T. , Vrionis Helen A. , Ortiz-Bernad Irene , Resch Charles T. , Long Philip E. , Dayvault Richard , Karp Ken , Marutzky Sam , Metzler Donald R. , Peacock Aaron , White David C. , Lowe Mary , Lovley Derek R.
Published: October 2003

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 μM in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of
Geobacter
species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and
Geobacter
species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of
Geobacter
species.
Effects of Iron Limitation on Adherence and Cell Surface Carbohydrates of
Corynebacterium diphtheriae
Strains
Authors: Moreira Lílian de Oliveira , Andrade Arnaldo Feitosa Braga , Vale Márcio Damasceno , Souza Sônia Maria Silva , Hirata Raphael , Asad Lídia Maria Oliveira Buarque , Asad Nasser Ribeiro , Monteiro-Leal Luiz Henrique , Previato José Osvaldo , Mattos-Guaraldi Ana Luiza
Published: October 2003

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of
Corynebacterium diphtheriae
subsp.
mitis
and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl
3
. The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in
d
-mannose and
d
-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of
C. diphtheriae
surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for
d
-mannose,
d
-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For
C. diphtheriae
, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.
Role of Ectoine in
Vibrio cholerae
Osmoadaptation
Authors: Pflughoeft Kathryn J. , Kierek Katharine , Watnick Paula I.
Published: October 2003

Vibrio cholerae
is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment,
V. cholerae
must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of
V. cholerae
to increases in environmental osmolarity. Optimal growth of
V. cholerae
in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted
V. cholerae
displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted
V. cholerae
displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for
V. cholerae
population dynamics when seawater and freshwater and their attendant microbes mix.
Population Dynamics of
Vibrio fischeri
during Infection of
Euprymna scolopes
Authors: McCann Jessica , Stabb Eric V. , Millikan Deborah S. , Ruby Edward G.
Published: October 2003

The luminous bacterium
Vibrio fischeri
colonizes a specialized light-emitting organ within its squid host,
Euprymna scolopes
. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of
V. fischeri
during colonization more fully, we used mini-Tn
7
transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on
V. fischeri
cells carrying the transposon, which inserts in single copy in a specific intergenic region of the
V. fischeri
genome. Strains marked with mini-Tn
7
also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of
V. fischeri
cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of
V. fischeri
were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of
V. fischeri
, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.
Tri16Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production byFusarium sporotrichioides
Authors: Peplow Andrew W. , Meek Isaac B. , Wiles Melinda C. , Phillips Timothy D. , Beremand Marian N.
Published: October 2003

We previously characterizedTri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis inFusarium sporotrichioidesNRRL 3299. Sequence analysis of the region surroundingTri1revealed a gene, namedTri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption ofTri16were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate thatTri16encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of aTri16ortholog inGibberella pulicarisR-6380 that is likely linked to a presumably inactive ortholog forTri1.
Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and
Pseudomonas aeruginosa
PAO1
Authors: Huang Jean J. , Han Jong-In , Zhang Lian-Hui , Leadbetter Jared R.
Published: October 2003

Acyl-homoserine lactones (AHLs) are employed by several
Proteobacteria
as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium,
Pseudomonas
strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of
Pseudomonas aeruginosa
, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium
P. aeruginosa
, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A,
P. aeruginosa
strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A
P. aeruginosa
gene,
pvdQ
(PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a
Ralstonia
species.
Escherichia coli
expressing
pvdQ
catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL.
P. aeruginosa
engineered to constitutively express
pvdQ
did not accumulate its 3OC12HSL quorum signal when grown in rich media. However,
pvdQ
knockout mutants of
P. aeruginosa
were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other γ-
Proteobacteria
, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.
Comparing Denitrification Estimates for a Texas Estuary by Using Acetylene Inhibition and Membrane Inlet Mass Spectrometry
Authors: Bernot Melody J. , Dodds Walter K. , Gardner Wayne S. , McCarthy Mark J. , Sobolev Dmitri , Tank Jennifer L.
Published: October 2003

Characterizing denitrification rates in aquatic ecosystems is essential to understanding how systems may respond to increased nutrient loading. Thus, it is important to ensure the precision and accuracy of the methods employed for measuring denitrification rates. The acetylene (C2H2) inhibition method is a simple technique for estimating denitrification. However, potential problems, such as inhibition of nitrification and incomplete inhibition of nitrous oxide reduction, may influence rate estimates. Recently, membrane inlet mass spectrometry (MIMS) has been used to measure denitrification in aquatic systems. Comparable results were obtained with MIMS and C2H2inhibition methods when chloramphenicol was added to C2H2inhibition assay mixtures to inhibit new synthesis of denitrifying enzymes. Dissolved-oxygen profiles indicated that surface layers of sediment cores subjected to the MIMS flowthrough incubation remained oxic whereas cores incubated using the C2H2inhibition methods did not. Analysis of the microbial assemblages before and after incubations indicated significant changes in the sediment surface populations during the long flowthrough incubation for MIMS analysis but not during the shorter incubation used for the C2H2inhibition method. However, bacterial community changes were also small in MIMS cores at the oxygen transition zone where denitrification occurs. The C2H2inhibition method with chloramphenicol addition, conducted over short incubation intervals, provides a cost-effective method for estimating denitrification, and rate estimates are comparable to those obtained by the MIMS method.
Bacterial Chemotaxis to Naphthalene Desorbing from a Nonaqueous Liquid
Authors: Law Aaron M. J. , Aitken Michael D.
Published: October 2003

Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined. We studied the effect of chemotaxis by
Pseudomonas putida
G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL. Chemotaxis by wild-type
P. putida
G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains. While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source. The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation. The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.
Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils
Authors: Rich J. J. , Heichen R. S. , Bottomley P. J. , Cromack K. , Myrold D. D.
Published: October 2003

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers.
nosZ
, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of
nosZ
terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225
nosZ
clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of
nosZ
fragments sequenced from meadow or forest soils were most similar to
nosZ
from the
Rhizobiaceae
group in α-
Proteobacteria
species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.
Identification and Characterization of the
CYP52
Family of
Candida tropicalis
ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids
Authors: Craft David L. , Madduri Krishna M. , Eshoo Mark , Wilson C. Ron
Published: October 2003

Candida tropicalis
ATCC 20336 excretes α,ω-dicarboxylic acids as a by-product when cultured on
n
-alkanes or fatty acids as the carbon source. Previously, a β-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the ω-hydroxylase enzymes catalyzing the first step in the ω-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of ω-oxidation of
n
-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (
CYP52
) of
C. tropicalis
ATCC20336 as well as the accompanying
NCP
were cloned and sequenced. The 10
CYP
genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes,
CYP52A13
and
CYP52A14
showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair
CYP52A17
and
CYP52A18
was also induced under all three conditions but to a lesser extent. Moderate induction of
CYP52A12
was observed. These results identify the
CYP52
and
NCP
genes as being involved in α,ω-dicarboxylic acid production by
C. tropicalis
and provide the foundation for biocatalyst improvement.
Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of
Candida tropicalis
Authors: Eschenfeldt William H. , Zhang Yeyan , Samaha Hend , Stols Lucy , Eirich L. Dudley , Wilson C. Ronald , Donnelly Mark I.
Published: October 2003

Candida tropicalis
ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase.
C. tropicalis
ATCC 20336 contains a family of
CYP
genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C
18:1
), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the
C. tropicalis
cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C
14:0
) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.
Distribution of the Coenzyme M Pathway of Epoxide Metabolism among Ethene- and Vinyl Chloride-Degrading
Mycobacterium
Strains
Authors: Coleman Nicholas V. , Spain Jim C.
Published: October 2003

An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of
Mycobacterium
strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from
Mycobacterium
strain JS60 and the propene oxidizers
Xanthobacter
strain Py2 and
Rhodococcus
strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The
Mycobacterium
EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of
Rhodococcus
strain B-276 than that of
Xanthobacter
strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the
Mycobacterium
strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.
Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of
N
ε
-Acetyl-β-Lysine and Growth at High Salinity
Authors: Pflüger K. , Baumann S. , Gottschalk G. , Lin W. , Santos H. , Müller V.
Published: October 2003

The compatible solute
N
ε
-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of
N
ε
-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (
ablA
) and β-lysine acetyltransferase (
ablB
), which are assumed to catalyze
N
ε
-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea
Methanosarcina mazei
Gö1,
Methanosarcina acetivorans
,
Methanosarcina barkeri
,
Methanococcus jannaschii
, and
Methanococcus maripaludis
. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the
abl
operon) in
M. mazei
Gö1. Expression of the
abl
operon was strictly salt dependent. The
abl
operon was deleted in the genetically tractable
M. maripaludis
. Δ
abl
mutants of
M. maripaludis
no longer produced
N
ε
-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the
abl
operon is essential for
N
ε
-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.
Optimized Expression of a Thermostable Xylanase from
Thermomyces lanuginosus
in
Pichia pastoris
Authors: Damaso Mônica C. Triches , Almeida Marcius S. , Kurtenbach Eleonora , Martins Orlando B. , Pereira Nei , Andrade Carolina M. M. C. , Albano Rodolpho M.
Published: October 2003

Highly efficient production of a
Thermomyces lanuginosus
IOC-4145 β-1,4-xylanase was achieved in
Pichia pastoris
under the control of the AOX1 promoter.
P. pastoris
colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 2
3
factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in
P. pastoris
makes this expression system attractive for fermentor growth and industrial applications.
Diversity ofWolbachiaEndosymbionts in Heteropteran Bugs
Authors: Kikuchi Yoshitomo , Fukatsu Takema
Published: October 2003

An extensive survey ofWolbachiaendosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers forwspandftsZgenes ofWolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infectingWolbachiatypes, and molecular phylogenetic characterization of all the detectedWolbachiastrains. Of 134 heteropteran species from 19 families examined,Wolbachiainfection was detected in 47 species from 13 families. From the 47 species, 59Wolbachiastrains were identified. Of the 59 strains, 16 and 43 were assigned to A group and B group in theWolbachiaphylogeny, respectively. The 47 species ofWolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection. Molecular phylogenetic analysis showed little congruence betweenWolbachiaphylogeny and host systematics, suggesting frequent horizontal transfers ofWolbachiain the evolutionary course of the Heteroptera. The phylogenetic analysis also revealed several novel lineages ofWolbachia. Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfectingWolbachiastrains are generally not intense and thatWolbachiacoinfections have been established through a stochastic process probably depending on occasional horizontal transfers.
Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by
Gluconacetobacter xylinus
I-2281
Authors: Kornmann Henri , Duboc Philippe , Marison Ian , von Stockar Urs
Published: October 2003

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain
Gluconacetobacter xylinus
I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by
G. xylinus.
In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing
G. xylinus
on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.
Molecular Analysis of the
rfb
O Antigen Gene Cluster of
Salmonella enterica
Serogroup O:6,14 and Development of a Serogroup-Specific PCR Assay
Authors: Fitzgerald Collette , Sherwood Rachel , Gheesling Linda L. , Brenner Frances W. , Fields Patricia I.
Published: October 2003

The Kauffmann-White scheme for serotyping
Salmonella
recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of
Salmonella
, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in
Salmonella.
The majority of the enzymes involved in O antigen biosynthesis are encoded by the
rfb
gene cluster. We report the sequencing of the
rfb
region from
S. enterica
serotype Sundsvall (serogroup O:6,14). The
S. enterica
serotype Sundsvall
rfb
region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized
rfb
regions, the serogroup O:6,14 sequence is most related to serogroup C
1
. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (
S. enterica
serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the
rfb
gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative
wzx
(O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of
Salmonella
serogroup O:6,14.