bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved nucleation of TiN atomic layer deposition films on SiLK low-k polymer dielectric using an Al2O3 atomic layer deposition adhesion layer
Authors: Elam J. W. , Wilson C. A. , Schuisky M. , Sechrist Z. A. , George S. M.
Published: June 2003

Diffusion barriers are required to prevent copper from diffusing into low-k polymer dielectrics in backend interconnects. The ability to deposit conformal diffusion barriers onto high aspect ratio, low-k polymer features requires atomic layer deposition (ALD) techniques. This study examined TiN ALD on SiLK (a trademark of the Dow Chemical Company) low-k polymer dielectric using tetrakis-dimethylamino titanium and NH3. X-ray fluorescence spectroscopy (XRFS), optical microscopy, and surface profiling of the TiN ALD films deposited on SiLK revealed discontinuous films displaying distinct patchy regions. The patches corresponded to a thinner TiN coating and were attributed to difficulties for TiN ALD nucleation on SiLK. To study TiN ALD nucleation, in situ quartz-crystal microbalance (QCM) measurements were performed by spincoating SiLK onto the QCM sensor. Subsequent QCM measurements during TiN ALD revealed very low initial TiN ALD growth rates indicating poor nucleation. Al2O3 ALD was then performed on the SiLK film using trimethyl aluminum and H2O. Surface profiling, XRFS, QCM, and transmission electron microscopy measurements revealed that the Al2O3 ALD film nucleates immediately on SiLK producing a continuous Al2O3 film. In addition, QCM measurements showed that TiN ALD nucleates readily on the Al2O3 surface. The Al2O3 ALD adhesion layer facilitated the growth of a continuous TiN ALD film on SiLK. Examination of TiN ALD films prepared on SiLK with progressively thinner Al2O3 ALD adhesion layers revealed that 10 Al2O3 ALD cycles were sufficient to promote the nucleation of the TiN ALD film.
X-ray photoelectron spectroscopy study of electrodeposited nanostructured CeO2 films
Authors: Wang Adele Qi , Punchaipetch Prakaipetch , Wallace Robert M. , Golden Teresa Diane
Published: June 2003

Nanostructured CeO2 films are produced by anodic electrodeposition onto a variety of substrates. The crystal structure and oxidation state of the films are studied by x-ray diffraction (XRD) and x-ray photoelectron spectroscopy (XPS), respectively. Calculations from XRD show that crystallite sizes of the electrodeposited films range from 6–10 nm. Sintering of these films to 700 °C increases the grain size to approximately 25 nm. A study of Ce 3d, Ce 4d, O 1s, and the valence-band region indicates that the Ce(IV)/Ce(III) ratio increases with sintering temperature, with features of both Ce4+ and Ce3+ identified by XPS. Ce 3d and O 1s characteristics show that high-temperature sintering of the films facilitates Ce(IV) oxide formation.
Rutherford backscattering analysis of GaN decomposition
Authors: Choi H. W. , Cheong M. G. , Rana M. A. , Chua S. J. , Osipowicz T. , Pan J. S.
Published: June 2003

The decomposition of GaN at temperatures ranging from 500 °C to 1100 °C has been studied by Rutherford backscattering (RBS), x-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The development of a surface defect peak is a consequence of preferential N2 loss at elevated temperatures. Additionally, broadening of the defect peak at 1100 °C, corresponding to a damage depth of approximately 0.25 μm beneath the surface, can be attributed to the diffusion of defects from the interface. At such temperatures, severe roughening of the surface is observed through AFM scans, which also correlated well with the damage depths estimated from RBS spectra. Nevertheless, Ga droplet formation is not detected from our samples as verified by XPS. Our results show that GaN remains thermally stable in N2 up to 900 °C. At higher temperatures, significant decomposition occurs and gives rise to degradation to the structural and morphological properties of the film.
Effect of chemical vapor deposition energy sources on the structure of SiC prepared by carbon nanotubes-confined reaction
Authors: Shajahan Md. , Mo Y. H. , Nahm K. S.
Published: June 2003

Silicon carbide (SiC) nanorods and microcrystals were synthesized with controlled structure through a two-step reaction scheme. Carbon nanotubes (CNTs) were first grown by the thermal decomposition of C2H2 over a Fe/Co metal catalyst loaded on a Si(100) wafer surface. The grown CNTs were then reacted with tetramethylsilane (TMS) to form β-SiC nanorods or microcrystals. SiC nanorods were observed when CNTs reacted with TMS at 1100 °C in an IR-induced heating chemical vapor deposition (CVD) reactor, whereas SiC microcrystals were formed at 1250 °C in an rf-induced-heating CVD reactor. Higher temperature and longer time were favorable for the growth of good quality SiC crystals. The blueshift of the PL spectra was observed from the SiC nanorods and SiC microcrystals. The structural change of CNTs in the growth was discussed in this work.
Effect of current spreading on luminescence improvement in selectively oxidized AlGaInP light-emitting diodes
Authors: Nee Tzer-En , Chien Kuo-Tai , Chou Yi-Lun , Chou Li-Chang , Lin Chung-Han , Lin Ray-Ming , Fang Bor-Ren , Chang Shi-Shya
Published: June 2003

We have systematically investigated the luminescence performance of AlGaInP light-emitting diodes with a wet-oxidized AlAs/AlGaAs distributed Bragg reflector structure. The peak intensity for a 20-μm-radius aperture is 4.2 times greater than that for a 100-μm-radius aperture at the same current density. We believe that this significant improvement in the emission characteristics with the Al-based native oxides is the result of the current-spreading effect. With an increase in the lateral depth of the oxidized layer, the near-field patterns become more centralized. This is attributed to the confinement of the current to the region about the unoxidized aperture. We also found that the centralized carriers contribute to the enhancement of the external quantum efficiency per unit area up to factor of 58.6.
Characteristics of plasma display with rf microdischarge
Authors: Kang J.
Published: June 2003

A plasma display utilizing a radio frequency (rf) microdischarge has been proposed and investigated with respect to its discharge characteristics and display performances. In order to generate a low electric field between electrodes, a relatively large spacing of 1 mm and a high driving frequency of 40 MHz were selected. A 4 in. prototype plasma display with red-green-blue phosphors was fabricated. Except for the fabrication of the barrier rib, conventional manufacturing processes used in ac plasma display panel were applied to fabricate a test panel. The white luminance was over 2000 cd/m2 in a Ne-5% Xe composition at 400 Torr. The efficiency of the panel was over 4 lm/W, which is comparable to that of a typical cathode-ray tube. The panel also showed good color purity due to the absence of Ne emission. A 36 gray level image was demonstrated using an address and rf sustained-period separated driving scheme.
Dynamics of Microbial Communities on Marine Snow Aggregates: Colonization, Growth, Detachment, and Grazing Mortality of Attached Bacteria
Authors: Kiørboe Thomas , Tang Kam , Grossart Hans-Peter , Ploug Helle
Published: June 2003

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm3h−1for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 103to 104and ∼102cells sphere−1, respectively, whereas bacterial populations increased at a declining rate to >107cells sphere−1. Attached microorganisms initially detached at high specific rates of ∼10−2min−1, but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day−1) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate−1h−1. At low bacterial densities, the flagellate surface clearance rate was ∼5 × 10−7cm2min−1, but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.
Identification and Characterization of Coenzyme B
12
-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures
Authors: Knietsch Anja , Bowien Susanne , Whited Gregg , Gottschalk Gerhard , Daniel Rolf
Published: June 2003

To isolate genes encoding coenzyme B
12
-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative
Escherichia coli
strain. In this way, two positive
E. coli
clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000
E. coli
clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of
Citrobacter freundii
and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from
E. coli
Bl21/pAK2.1 and
E. coli
Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from
E. coli
Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of
C. freundii
. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of
C. freundii
with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by
E. coli
Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.
Increased Production of Folate by Metabolic Engineering of
Lactococcus lactis
Authors: Sybesma Wilbert , Starrenburg Marjo , Kleerebezem Michiel , Mierau Igor , de Vos Willem M. , Hugenholtz Jeroen
Published: June 2003

The dairy starter bacterium
Lactococcus lactis
is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in
L. lactis
MG1363:
folA
,
folB
,
folKE
,
folP
, and
folC
. The gene
folKE
encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of
folKE
in
L. lactis
was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of
folKE
and
folC
, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of
folA
, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.
Mycotoxigenic
Fusarium
and Deoxynivalenol Production Repress Chitinase Gene Expression in the Biocontrol Agent
Trichoderma atroviride
P1
Authors: Lutz Matthias P. , Feichtinger Georg , Défago Geneviève , Duffy Brion
Published: June 2003

Mycotoxin contamination associated with head blight of wheat and other grains caused by
Fusarium culmorum
and
F. graminearum
is a chronic threat to crop, human, and animal health throughout the world. One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals. Certain
Fusarium
toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology
87:
1250-1257, 1997). The potential impact of DON on
Fusarium
competition with other microorganisms has not been described previously. Any competitive advantage conferred by DON would complicate efforts to control
Fusarium
during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings. We examined the effect of the DON mycotoxin on ecological interactions between pathogenic
Fusarium
and
Trichoderma atroviride
strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases. Expression of the
Trichoderma
chitinase genes,
ech42
and
nag1
, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using
goxA
reporter gene fusions. We found that DON-producing
F. culmorum
and
F. graminearum
strains repressed expression of
nag1-gox
. DON-negative wild-type
Fusarium
strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene,
tri5
, had no effect on antagonist gene expression. The role of DON as the principal repressor above other pathogen factors was confirmed. Exposure of
Trichoderma
to synthetic DON or to a non-DON-producing
Fusarium
mutant resulted in the same level of
nag1-gox
repression as the level observed with DON-producing
Fusarium
. DON repression was specific for
nag1-gox
and had no effect, either positive or negative, on expression of another key chitinase gene,
ech42.
This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in
Fusarium
competitiveness.
Characterization of the Microbial Community in Indoor Environments: a Chemical-Analytical Approach
Authors: Sebastian Aleksandra , Larsson Lennart
Published: June 2003

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of
13
C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.
Repeated Introduction of Genetically ModifiedPseudomonas putidaWCS358r without Intensified Effects on the Indigenous Microflora of Field-Grown Wheat
Authors: Viebahn M. , Glandorf D. C. M. , Ouwens T. W. M. , Smit E. , Leeflang P. , Wernars K. , Thomashow L. S. , van Loon L. C. , Bakker P. A. H. M.
Published: June 2003

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community,Pseudomonas putidaWCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 107CFU per g of roots soon after sowing to less than 102CFU per g after harvest 132 days after sowing. Thephzandphlgenes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.
Growth of Hyperthermophilic Archaeon
Pyrococcus furiosus
on Chitin Involves Two Family 18 Chitinases
Authors: Gao Jun , Bauer Michael W. , Shockley Keith R. , Pysz Marybeth A. , Kelly Robert M.
Published: June 2003

Pyrococcus furiosus
was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (
chiA
[Pf1234] and
chiB
[Pf1233]) were identified in the genome of
P. furiosus
, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA
BD
), that is related to binding domains associated with several previously studied bacterial chitinases.
chiB
, separated by 37 nucleotides from
chiA
and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB
BD
[11 kDa]), followed by a catalytic domain (ChiB
cat
[35 kDa]). No apparent signal peptide is encoded within
chiB
. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in
Escherichia coli.
ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for
P. furiosus
growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.
Ammonia-Oxidizing Bacteria along Meadow-to-Forest Transects in the Oregon Cascade Mountains
Authors: Mintie A. T. , Heichen R. S. , Cromack, K. , Myrold D. D. , Bottomley P. J.
Published: June 2003

Although nitrification has been well studied in coniferous forests of Western North America, communities of NH3-oxidizing bacteria in these forests have not been characterized. Studies were conducted along meadow-to-forest transects at two sites (Lookout and Carpenter) in the H. J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon. Soil samples taken at 10- or 20-m intervals along the transects showed that several soil properties, including net nitrogen mineralization and nitrification potential rates changed significantly between vegetation zones. Nonetheless, terminal restriction fragment length polymorphism (T-RFLP) analysis of the PCR-amplified NH3monooxygenase subunit A gene (amoA) showed the same DNA fragments (TaqI [283 bp],CfoI [66 bp], andAluI [392 bp]) to dominate ≥45 of 47 soil samples recovered from both sites. Two fragments (491-bpAluI [AluI491] andCfoI135) were found more frequently in meadow and transition zone soil samples than in forest samples at both sites. At the Lookout site the combinationAluI491-CfoI135 was found primarily in meadow samples expressing the highest N mineralization rates. Four uniqueamoAsequences were identified among 15 isolates recovered into pure culture from various transect locations. Six isolates possessed the most common T-RFLPamoAfingerprint of the soil samples (TaqI283-AluI392-CfoI66), and theiramoAsequences shared 99.8% similarity with a cultured species,Nitrosospirasp.strain Ka4 (cluster 4). The other threeamoAsequences were most similar to sequences ofNitrosospirasp.strain Nsp1 andNitrosospira briensis(cluster 3). 16S ribosomal DNA sequence analysis confirmed the affiliation of these isolates withNitrosospiraclusters 3 and 4. TwoamoAclone sequences matched T-RFLP fingerprints found in soil, but they were not found among the isolates.
Identification ofListeria monocytogenesGenes Involved in Salt and Alkaline-pH Tolerance
Authors: Gardan Rozenn , Cossart Pascale ,   The European Listeria Genome Consortium, Labadie Jean
Published: June 2003

The capacity ofListeria monocytogenesto tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZinsertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.
Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning inMicromonosporasp. Strain 40027
Authors: Li Xiaohua , Zhou Xiufen , Deng Zixin
Published: June 2003

Vector systems allowing autonomous or site-specific integrative gene cloning were developed forMicromonosporasp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derivedEscherichia coliplasmid vector, pSET152, mediated by theattP site of theStreptomycesphage ΦC31. Both vectors can be transferred by conjugation fromE. coliintoMicromonosporasp. strain 40027. The heterologous cloning and expression of thedndgene cluster originating fromStreptomyces lividans1326 intoMicromonosporasp. strain 40027 demonstrated the use of the two systems.
Aerobic Denitrifying Bacteria That Produce Low Levels of Nitrous Oxide
Authors: Takaya Naoki , Catalan-Sakairi Maria Antonina B. , Sakaguchi Yasushi , Kato Isao , Zhou Zhemin , Shoun Hirofumi
Published: June 2003

Most denitrifiers produce nitrous oxide (N
2
O) instead of dinitrogen (N
2
) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N
2
O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO
3
−
) to N
2
at rates of 0.9 and 0.03 μmol min
−1
unit of optical density at 540 nm
−1
at dissolved oxygen (O
2
) (DO) concentrations of 39 and 38 μmol liter
−1
, respectively. At the same DO level, the typical denitrifier
Pseudomonas stutzeri
and the previously described aerobic denitrifier
Paracoccus denitrificans
did not produce N
2
but evolved more than 10-fold more N
2
O than strains TR2 and K50 evolved. The isolates denitrified NO
3
−
with concomitant consumption of O
2
. These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the β subclass of the class
Proteobacteria
and were identified as
P. stutzeri
TR2 and
Pseudomonas
sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N
2
O emission, the single-stage process for nitrogen removal, and microbial N
2
O emission into the ecosystem.
A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater
Authors: Fout G. Shay , Martinson Beth C. , Moyer Michael W. N. , Dahling Daniel R.
Published: June 2003

Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.
Identification of Intermediate and Branch Metabolites Resulting from Biotransformation of 2-Benzoxazolinone by
Fusarium verticillioides
Authors: Glenn A. E. , Meredith F. I. , Morrison W. H. , Bacon C. W.
Published: June 2003

Detoxification of the maize (
Zea mays
) antimicrobial compound 2-benzoxazolinone by the fungal endophyte
Fusarium verticillioides
involves two genetic loci,
FDB1
and
FDB2
, and results in the formation of
N
-(2-hydroxyphenyl)malonamic acid. Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway. Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X. Overall, 2-benzoxazolinone metabolism involves hydrolysis (
FDB1
) to produce 2-aminophenol, which is then modified (
FDB2
) by addition of a malonyl group to produce
N
-(2-hydroxyphenyl)malonamic acid. If the modification is prevented due to genetic mutation (
fbd2
), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol. This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce
N
-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications. Detoxification of these antimicrobials by
F. verticillioides
apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris.
Intraspecific Diversity of
Vibrio vulnificus
in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR
Authors: Lin Meilan , Payne Deborah A. , Schwarz John R.
Published: June 2003

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208
Vibrio vulnificus
strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001.
V. vulnificus
was not detected during the winter months (December through February). The densities of
V. vulnificus
in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208
V. vulnificus
isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of
V. vulnificus
strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among
V. vulnificus
isolates. These results suggest that a great genetic diversity of
V. vulnificus
strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.
Bacteriophage Ecology in Commercial Sauerkraut Fermentations
Authors: Lu Z. , Breidt F. , Plengvidhya V. , Fleming H. P.
Published: June 2003

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included
Leuconostoc
,
Weissella
, and
Lactobacillus
species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family
Myoviridae
or
Siphoviridae
and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two
Lactobacillus
species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.
Population Genetics of
Vibrio vulnificus
: Identification of Two Divisions and a Distinct Eel-Pathogenic Clone
Authors: Gutacker Michaela , Conza Nadine , Benagli Cinzia , Pedroli Ambra , Bernasconi Marco Valerio , Permin Lise , Aznar Rosa , Piffaretti Jean-Claude
Published: June 2003

Genetic relationships among 62
Vibrio vulnificus
strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the
recA
and
glnA
genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the
V. vulnificus
population into two divisions (divisions Ι and ΙΙ). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62
V. vulnificus
isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the
recA
gene and of 402 bp of the
glnA
gene also separated the
V. vulnificus
population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the
V. vulnificus
population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions Ι and ΙΙ) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.
Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors
Authors: Egli Konrad , Langer Christian , Siegrist Hans-Ruedi , Zehnder Alexander J. B. , Wagner Michael , van der Meer Jan Roelof
Published: June 2003

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus
Nitrospira
were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the
amoA
gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the
Nitrosomonas communis
and
Nitrosomonas oligotropha
sublineages and the
Nitrosomonas europaea-Nitrosomonas eutropha
subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one
amoA
RFLP type from the
N. europaea-N. eutropha
group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in
amoA
RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day
−1
compared to 0.2 day
−1
for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.
Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil
Authors: Ellis Richard J. , Morgan Philip , Weightman Andrew J. , Fry John C.
Published: June 2003

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.
Unusual Organization for Lactose and Galactose Gene Clusters in
Lactobacillus helveticus
Authors: Fortina Maria Grazia , Ricci Giovanni , Mora Diego , Guglielmetti Simone , Manachini Pier Luigi
Published: June 2003

The nucleotide sequences of the
Lactobacillus helveticus
lactose utilization genes were determined, and these genes were located and oriented relative to one another. The
lacLM
genes (encoding the β-galactosidase protein) were in a divergent orientation compared to
lacR
(regulatory gene) and
lacS
(lactose transporter). Downstream from
lacM
was an open reading frame (
galE
) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as
lacM
. The
lacR
gene was separated from the downstream
lacS
gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (
lacS
′). Northern blot analysis revealed that
lacL
,
lacM
, and
galE
made up an operon that was transcribed in the presence of lactose from an upstream
lacL
promoter. The inducible genes
lacL
and
lacM
were regulated at the transcriptional level by the LacR repressor. In the presence of glucose and galactose
galE
was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively. Lactose transport was inducible by addition of lactose to the growth medium.
Involvement of the Reserve Material Poly-β-Hydroxybutyrate inAzospirillum brasilenseStress Endurance and Root Colonization
Authors: Kadouri Daniel , Jurkevitch Edouard , Okon Yaacov
Published: June 2003

When grown under suboptimal conditions, rhizobacteria of the genusAzospirillumproduce high levels of poly-β-hydroxybutyrate (PHB).Azospirillum brasilensestrain Sp7 and aphbC(PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source byA. brasilenseunder stress conditions favor establishment of this bacterium and its survival in competitive environments. However, inA. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.
Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in
Saccharomyces cerevisiae
Authors: Thomsson Elisabeth , Larsson Christer , Albers Eva , Nilsson Annika , Franzén Carl Johan , Gustafsson Lena
Published: June 2003

Seven different strains of
Saccharomyces cerevisiae
were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.
External pH and Nitrogen Source Affect Secretion of Pectate Lyase by
Colletotrichum gloeosporioides
Authors: Drori N. , Kramer-Haimovich H. , Rollins J. , Dinoor A. , Okon Y. , Pines O. , Prusky D.
Published: June 2003

Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by
Colletotrichumgloeosporioides.
As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither
pelB
(encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased. Nitrogen assimilation also was required for PL secretion at pH 6.0. Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0. Sequence analysis of the 5′ upstream region of the
pelB
promoter revealed nine putative consensus binding sites for the
Aspergillus
transcription factor PacC. Consistent with this result, the transcript levels of
pac1
(the
C. gloeosporioides pacC
homologue) and
pelB
increased in parallel as a function of pH. Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of
C. gloeosporioides
.
Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading
Pseudomonas putida
Cg1
Authors: Park Woojun , Jeon Che Ok , Hohnstock-Ashe Amy M. , Winans Stephen C. , Zylstra Gerben J. , Madsen Eugene L.
Published: June 2003

Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by
Pseudomonas putida
Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in
P. putida
NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (
mpf
) gene,
mpfA
encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other
trb
-like
mpf
genes. Sequence comparison revealed that the 10
mpf
genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative
mpf
genes of catabolic plasmid pWW0 (TOL plasmid of
P. putida
) and pM3 (antibiotic resistance plasmid of
Pseudomonas.
spp). A polar mutation caused by insertional inactivation in
mpfA
of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this
mpf
region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to
mpfA. lacZ
transcriptional fusions revealed that
mpf
genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in
P. putida
Cg1 was enhanced by expression of genes in the
mpf
region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.