bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Structural and Physiological Profiles To Assess the Effects of Cu on Biofilm Microbial Communities
Authors: Massieux B. , Boivin M. E. Y. , van den Ende F. P. , Langenskiöld J. , Marvan P. , Barranguet C. , Admiraal W. , Laanbroek H. J. , Zwart G.
Published: August 2004

We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 μM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.
Inactivation of Caliciviruses
Authors: Duizer Erwin , Bijkerk Paul , Rockx Barry , de Groot Astrid , Twisk Fleur , Koopmans Marion
Published: August 2004

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100°C. UV-B radiation from 0 to 150 mJ/cm
2
caused dose-dependent inactivation, with a 3
D
(
D
= 1 log
10
) reduction in infectivity at 34 mJ/cm
2
for both viruses. Inactivation by 70% ethanol was inefficient, with only 3
D
reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.
Oxidation of Methyl
tert
-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and
n
-Octane-Grown
Pseudomonas putida
GPo1
Authors: Smith Christy A. , Hyman Michael R.
Published: August 2004

The alkane hydroxylase enzyme system in
Pseudomonas putida
GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl
tert
-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of
tert
-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized
tert
-amyl methyl ether but did not appear to oxidize either TBA, ethyl
tert
-butyl ether, or
tert
-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of
P. putida
GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all
n
-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting
n
-alkanes (propane and
n
-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in
n
-octane-grown cells. Kinetic studies with strain GPo1 grown on
n
-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in
n
-octane-grown cells because of the unusually high

K
s

value (20 to 40 mM) for MTBE.
Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in
Streptococcus gordonii
Authors: Lee Chiang W. , Lee Song F. , Halperin Scott A.
Published: August 2004

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in
Streptococcus gordonii
DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant
S. gordonii
recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant
S. gordonii
expressing double copies of DTA (SpaP-DTA
2
) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response.
S. gordonii
SpaP-DTA
2
was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.
Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR
Authors: Kostal Jan , Yang Rosanna , Wu Cindy H. , Mulchandani Ashok , Chen Wilfred
Published: August 2004

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in
Escherichia coli
in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd
2+
or Zn
2+
accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.
Characterization of a Galactokinase-Positive Recombinant Strain of
Streptococcus thermophilus
Authors: Vaillancourt Katy , LeMay Jean-Dominique , Lamoureux Maryse , Frenette Michel , Moineau Sylvain , Vadeboncoeur Christian
Published: August 2004

The lactic acid bacterium
Streptococcus thermophilus
is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species
Streptococcus salivarius
,
S. thermophilus
is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of
S. thermophilus
to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal
−
strain
S. thermophilus
SMQ-301 with
S. salivarius galK
, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing
S. salivarius galK
, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of
S. salivarius galK
and (ii) transcription of the plasmid-borne
galK
gene did not require GalR, a transcriptional regulator of the
gal
and
lac
operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike
S. salivarius
, the ability to metabolize galactose was not sufficient for
S. thermophilus
to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal
+
strain grew and produced acid more rapidly than the Gal
−
wild-type strain.
Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of
Bombyx mori
, That Binds to Cry1A Toxins
Authors: Hossain Delwar M. , Shitomi Yasuyuki , Moriyama Kenta , Higuchi Masahiro , Hayakawa Tohru , Mitsui Toshiaki , Sato Ryoichi , Hori Hidetaka
Published: August 2004

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of
Bombyx mori
. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and

K
d

constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.
Development and Evaluation of an Online CO
2
Evolution Test and a Multicomponent Biodegradation Test System
Authors: Strotmann Uwe , Reuschenbach Peter , Schwarz Helmut , Pagga Udo
Published: August 2004

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO
2
) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO
2
method with existing CO
2
evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO
2
evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO
2
and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.
Mining the Microbiota of the Neonatal Gastrointestinal Tract for Conjugated Linoleic Acid-Producing Bifidobacteria
Authors: Rosberg-Cody E. , Ross R. P. , Hussey S. , Ryan C. A. , Murphy B. P. , Fitzgerald G. F. , Devery R. , Stanton C.
Published: August 2004

This study was designed to isolate different strains of the genus
Bifidobacterium
from the fecal material of neonates and to assess their ability to produce the
cis
-9,
trans
-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be
Bifidobacterium
species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml
−1
by using gas-liquid chromatography. The most efficient producers belonged to the species
Bifidobacterium breve
, of which two different strains converted 29 and 27% of the free linoleic acid to the
cis
-9,
trans
-11 isomer per microgram of dry cells, respectively. In addition, a strain of
Bifidobacterium bifidum
showed a conversion rate of 18%/μg dry cells. The ability of some
Bifidobacterium
strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.
Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag
Authors: Moldes Cristina , García José L. , García Pedro
Published: August 2004

The thermophilic inorganic pyrophosphatase (Pyr) fromThermus thermophilushas been produced inEscherichia colifused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.
Genetic Diversity of
Escherichia coli
Isolated from Urban Rivers and Beach Water
Authors: McLellan Sandra L.
Published: August 2004

Repetitive element anchored PCR was used to evaluate the genetic profiles of
Escherichia coli
isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of
E. coli
obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain
E. coli
strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained
E. coli
most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.
Effect of Nematodes on Rhizosphere Colonization by Seed-Applied Bacteria
Authors: Knox Oliver G. G. , Killham Ken , Artz Rebekka R. E. , Mullins Chris , Wilson Michael
Published: August 2004

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied
Pseudomonas fluorescens
SBW25 was capable of rhizosphere colonization at matric potentials of −10 and −40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.
Mutational Analysis of Mesentericin Y105, an Anti-
Listeria
Bacteriocin, for Determination of Impact on Bactericidal Activity, In Vitro Secondary Structure, and Membrane Interaction
Authors: Morisset Dany , Berjeaud Jean-Marc , Marion Didier , Lacombe Christian , Frère Jacques
Published: August 2004

Mesentericin Y105 is a 37-residue bacteriocin produced by
Leuconostoc mesenteroides
Y105 that displays antagonistic activity against gram-positive bacteria such as
Enterococcus faecalis
and
Listeria monocytogenes
. It is closely related to leucocin A, an antimicrobial peptide containing β-sheet and α-helical structures. To analyze structure-function relationships and the mode of action of this bacteriocin, we generated a collection of mesentericin derivatives. Mutations were obtained mostly by PCR random mutagenesis, and the peptides were produced by an original system of heterologous expression recently described (D. Morisset and J. Frère, Biochimie 84:569-576, 2002). Ten derivatives were obtained displaying modifications at eight different positions in the mesentericin Y105 sequence. Purified peptides were incorporated into lysophosphatidylcholine micelles and analyzed by circular dichroism. The α-helical contents of these peptides were compared and related to their respective bactericidal activities. Moreover, studies of the intrinsic fluorescence of tryptophan residues naturally occurring at positions 18 and 37 revealed information about insertion of the peptides in micelles. A model for the mode of action of mesentericin Y105 and related bacteriocins is proposed.
Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos
Authors: Cho Catherine Mee-Hie , Mulchandani Ashok , Chen Wilfred
Published: August 2004

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in thekcat/Kmvalue for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.
Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts
Authors: Ramírez Manuel , Vinagre Antonia , Ambrona Jesús , Molina Felipe , Maqueda Matilde , Rebollo José E.
Published: August 2004

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of
Saccharomyces cerevisiae
. Spontaneous

cyh2
R

/

cyh2
R

mutants resistant to high levels of cycloheximide can be directly isolated from

cyh2
S

/

cyh2
S

wine yeasts. Heterozygous

cyh2
R
/cyh2
S

hybrid clones vary in genetic instability as measured by loss of heterozygosity at
cyh2
. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become

cyh2
R

/

cyh2
R

homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.
Enhanced Anaerobic Biodegradation of Benzene-Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns
Authors: Da Silva Marcio L. B. , Alvarez Pedro J. J.
Published: August 2004

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and
o
-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of
bssA
gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential.
bssA
continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.
Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis
Authors: Buttner Mark P. , Cruz Patricia , Stetzenbach Linda D. , Klima-Comba Amy K. , Stevens Vanessa L. , Cronin Tracy D.
Published: August 2004

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of
Bacillus atrophaeus
(“
Bacillus subtilis
subsp.
niger
,” also referred to as BG), a
Bacillus anthracis
surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10
5
to 10
6
CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable
B. atrophaeus
spores were detected. After decontamination with chlorine dioxide gas, no culturable
B. atrophaeus
was detected in 24 of 27 samples (89%). However, QPCR analysis showed that
B. atrophaeus
DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.
Determining Rates of Change and Evaluating Group-Level Resiliency Differences in Hyporheic Microbial Communities in Response to Fluvial Heavy-Metal Deposition
Authors: Feris Kevin P. , Ramsey Philip W. , Rillig Matthias , Moore Johnnie N. , Gannon James E. , Holben William E.
Published: August 2004

Prior field studies by our group have demonstrated a relationship between fluvial deposition of heavy metals and hyporheic-zone microbial community structure. Here, we determined the rates of change in hyporheic microbial communities in response to heavy-metal contamination and assessed group-level differences in resiliency in response to heavy metals. A controlled laboratory study was performed using 20 flowthrough river mesocosms and a repeated-measurement factorial design. A single hyporheic microbial community was exposed to five different levels of an environmentally relevant metal treatment (0, 4, 8, 16, and 30% sterilized contaminated sediments). Community-level responses were monitored at 1, 2, 4, 8, and 12 weeks via denaturing gradient gel electrophoresis and quantitative PCR using group-specific primer sets for indigenous populations most closely related to the α-, β-, and γ-proteobacteria. There was a consistent, strong curvilinear relationship between community composition and heavy-metal contamination (
R
2
= 0.83;
P
< 0.001), which was evident after only 7 days of metal exposure (i.e., short-term response). The abundance of each phylogenetic group was negatively affected by the heavy-metal treatments; however, each group recovered from the metal treatments to a different extent and at a unique rate during the course of the experiment. The structure of hyporheic microbial communities responded rapidly and at contamination levels an order of magnitude lower than those shown to elicit a response in aquatic macroinvertebrate assemblages. These studies indicate that hyporheic microbial communities are a sensitive and useful indicator of heavy-metal contamination in streams.
Uptake of the β-Lactam Precursor α-Aminoadipic Acid in
Penicillium chrysogenum
Is Mediated by the Acidic and the General Amino Acid Permease
Authors: Trip Hein , Evers Melchior E. , Kiel Jan A. K. W. , Driessen Arnold J. M.
Published: August 2004

External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus
Penicillium chrysogenum
leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes,
PcGAP1
and
PcDIP5
, of
P. chrysogenum
were cloned and functionally expressed in a mutant of
Saccharomyces cerevisiae
(M4276) in which the acidic amino acid and general amino acid permease genes (
DIP5
and
GAP1
, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (

K
m

values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing
P. chrysogenum
. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.
Acid Resistance Systems Required for Survival ofEscherichia coliO157:H7 in the Bovine Gastrointestinal Tract and in Apple Cider Are Different
Authors: Price Stuart B. , Wright James C. , DeGraves Fred J. , Castanie-Cornet Marie-Pierre , Foster John W.
Published: August 2004

Escherichia coliO157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is σSdependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutantE. coliO157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate thatE. coliO157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.
Effects of Different Spices Used in Production of Fermented Sausages on Growth of and Curvacin A Production byLactobacillus curvatusLTH 1174
Authors: Verluyten Jurgen , Leroy Frédéric , de Vuyst Luc
Published: August 2004

Lactobacillus curvatusLTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production withL. curvatusLTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (Xmax) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increasedXmax. Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.
Overexpression of
Lactobacillus casei
d
-Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese
Authors: Broadbent Jeffery R. , Gummalla Sanjay , Hughes Joanne E. , Johnson Mark E. , Rankin Scott A. , Drake Mary Anne
Published: August 2004

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that α-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable α-keto acids to more-stable α-hydroxy acids via the action of α-keto acid dehydrogenases such as
d
-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the
Lactobacillus casei
d
-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into
L. casei
ATCC 334. Enzyme assays confirmed that α-keto acid dehydrogenase activity was significantly higher in pTRKH2:
dhic
transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with
Lactococcus lactis
starter only, starter plus
L. casei
ATCC 334, and starter plus
L. casei
ATCC 334 transformed with pTRKH2:
dhic
. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of
d
-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of α-keto acids, but sensory work indicated that this effect retarded cheese flavor development.
Influence of Humic Substances on Bacterial and Viral Dynamics in Freshwaters
Authors: Anesio Alexandre M. , Hollas Christin , Granéli Wilhelm , Laybourn-Parry Johanna
Published: August 2004

Bacterial and viral abundances were measured in 24 lakes with dissolved organic carbon (DOC) concentrations ranging from 3 to 19 mg of C liter−1. In addition, a laboratory experiment was performed to test the effects of different sources of carbon (i.e., glucose and fulvic acids) and nutrients on the dynamics of viruses and bacteria. In the lake survey, no correlation was found between virus abundance and DOC concentration, yet there was a significant positive correlation between bacterial abundance and DOC concentration. A negative correlation was found between the virus-to-bacteria ratio and DOC level. These results are in agreement with our findings in the laboratory, where virus counts were significantly lower in treatments with fulvic acid additions than in a control (mean, 67.4% ± 6.5% of the control). Virus counts did not differ significantly among the control and treatments with glucose, indicating that it was the type of organic carbon and not quantity which had an impact on viruses. Results from this study suggest that the way viruses control bacterial assemblages in humic lakes is different from the mechanism in clear water systems.
Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from
Candida tropicalis
Strain ATCC 20336
Authors: Eirich L. Dudley , Craft David L. , Steinberg Lisa , Asif Afreen , Eschenfeldt William H. , Stols Lucy , Donnelly Mark I. , Wilson C. Ron
Published: August 2004

Candida
tropicalis
(ATCC 20336) converts fatty acids to long-chain
dicarboxylic acids via a pathway that includes among other reactions
the oxidation of ω-hydroxy fatty acids to ω-aldehydes
by a fatty alcohol oxidase (FAO). Three
FAO
genes (one gene
designated
FAO1
and two putative allelic genes designated
FAO2a
and
FAO2b
), have been cloned and sequenced from
this strain. A comparison of the DNA sequence homology and derived
amino acid sequence homology between these three genes and previously
published
Candida FAO
genes indicates that
FAO1
and
FAO2
are distinct genes. Both genes were
individually cloned and expressed in
Escherichia coli
. The
substrate specificity and
K

m

values for
the recombinant FAO1 and FAO2 were significantly different.
Particularly striking is the fact that FAO1 oxidizes ω-hydroxy
fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but
not ω-hydroxy fatty acids. Analysis of extracts of strain H5343
during growth on fatty acids indicated that only FAO1 was highly
induced under these conditions.
FAO2
contains one CTG codon,
which codes for serine (amino acid 177) in
C. tropicalis
but
codes for leucine in
E. coli
. An
FAO2a
construct,
with a TCG codon (codes for serine in
E. coli
) substituted for
the CTG codon, was prepared and expressed in
E. coli
. Neither
the substrate specificity nor the
K

m

values for the FAO2a variant with a serine at position 177 were
radically different from those of the variant with a leucine at that
position.
Binary Toxins from
Bacillus thuringiensis
Active against the Western Corn Rootworm,
Diabrotica virgifera virgifera
LeConte
Authors: Baum James A. , Chu Chi-Rei , Rupar Mark , Brown Gregory R. , Donovan William P. , Huesing Joseph E. , Ilagan Oliver , Malvar Thomas M. , Pleau Michael , Walters Matthew , Vaughn Ty
Published: August 2004

The western corn rootworm,
Diabrotica virgifera virgifera
LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium
Bacillus thuringiensis
active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by
B. thuringiensis
strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.
Investigation of Spa Pools Associated with Lung Disorders Caused by
Mycobacterium avium
Complex in Immunocompetent Adults
Authors: Lumb Richard , Stapledon Richard , Scroop Andrew , Bond Peter , Cunliffe David , Goodwin Allan , Doyle Robyn , Bastian Ivan
Published: August 2004

Three cases of
Mycobacterium avium
complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations. Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of
M. avium
complex of 4.3 × 10
4
and 4.5 × 10
3
CFU/ml. Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated
M. avium
complex from the spa pool. A fourth case of
Mycobacterium avium
complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken. This spa pool contained low numbers of mycobacteria by smear and was culture positive for
M. avium
complex, and the nonmycobacterial organism count was 5.2 × 10
6
CFU/ml. Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers.
“
Candidatus
Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan
Bugula simplex
Authors: Lim Grace E. , Haygood Margo G.
Published: August 2004

The bryozoans
Bugula neritina
and
Bugula simplex
harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In
B. neritina
, the bacterial symbiont has been characterized as a gamma-proteobacterium, “
Candidatus
Endobugula sertula.” “
Candidatus
E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in
B. simplex
larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “
Candidatus
E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the
B. simplex
symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a
B. simplex
larva. The taxonomic status “
Candidatus
Endobugula glebosa” is proposed for the
B. simplex
larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from
B. simplex
that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with
B. simplex
. Bryostatin activity assays and chemical analyses of
B. simplex
extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in
B. simplex
that is similar and evolutionarily related to that in
B. neritina
.
Correspondence between Community Structure and Function during Succession in Phenol- and Phenol-plus-Trichloroethene-Fed Sequencing Batch Reactors
Authors: Ayala-del-Río Héctor L. , Callister Stephen J. , Criddle Craig S. , Tiedje James M.
Published: August 2004

The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors. One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day. After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg
−1
day
−1
for 2 years. In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg
−1
day
−1
. Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function. In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates. Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate). Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high. Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates. In contrast, the community under no stress exhibited a dynamic structure and dynamic function.