bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of Microorganisms in Fe-As-Rich Acid Mine Drainage Waters of Carnoulès, France
Authors: Bruneel O. , Duran R. , Casiot C. , Elbaz-Poulichet F. , Personné J.-C.
Published: January 2006

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg · liter
−1
), iron (750 to 2,700 mg · liter
−1
), and sulfate (2,000 to 7,500 mg · liter
−1
). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with
Gallionella ferruginea
, a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.
Persistence of
Mycobacterium avium
subsp.
paratuberculosis
and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure
Authors: Grewal Sukhbir K. , Rajeev Sreekumari , Sreevatsan Srinand , Michel Frederick C.
Published: January 2006

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as
Escherichia coli
O157:H7,
Listeria monocytogenes
,
Salmonella
spp., and
Mycobacterium avium
subsp.
paratuberculosis
(
Mycobacterium paratuberculosis
). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated
M. paratuberculosis
, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with
M. paratuberculosis
at 10
6
CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of
M. paratuberculosis
was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO
2
, while no significant change in carbon content was observed in the liquid slurry.
Escherichia coli
,
Salmonella
, and
Listeria
were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days.
M. paratuberculosis
was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56,
M. paratuberculosis
was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However,
M. paratuberculosis
DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.
Influence of Nutrient Inputs, Hexadecane, and Temporal Variations on Denitrification and Community Composition of River Biofilms
Authors: Chénier M. R. , Beaumier D. , Fortin N. , Roy R. , Driscoll B. T. , Lawrence J. R. , Greer C. W.
Published: January 2006

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001, supplemented with carbon (glucose), nitrogen (NH4Cl), phosphorus (KH2PO4), or combined nutrients (CNP), with or without hexadecane, a model compound representing aliphatic hydrocarbons used to simulate a pollutant. In fall 1999 and fall 2001, comparable denitrification activities and catabolic potentials were observed in the biofilms, implying that denitrifying populations showed similar activity patterns and catabolic potentials during the fall from year to year in this river ecosystem, when environmental conditions were similar. BothnirSandnirKdenitrification genes were detected by PCR amplification, suggesting that both denitrifying bacterial subpopulations can potentially contribute to total denitrification. Between 91.7 and 99.8% of the consumed N was emitted in the form of N2, suggesting that emission of N2O, a major potent greenhouse gas, by South Saskatchewan River biofilms is low. Denitrification was markedly stimulated by the addition of CNP, andnirSandnirKgenes were predominant only in the presence of CNP. In contrast, individual nutrients had no impact on denitrification and on the occurrence ofnirSandnirKgenes detected by PCR amplification. Similarly, only CNP resulted in significant increases in algal and bacterial biomass relative to control biofilms. Biomass measurements indicated a linkage between autotrophic and heterotrophic populations in the fall 1999 biofilms. Correlation analyses demonstrated a significant relationship (P≤ 0.05) between the denitrification rate and the biomass of algae and heterotrophic bacteria but not cyanobacteria. At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification in both years, slightly more in the fall of 2001. This study suggested that the response of the anaerobic heterotrophic biofilm community may be cyclic and predictable from year to year and that there are interactive effects between nutrients and the contaminant hexadecane.
Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in
Burkholderia xenovorans
LB400
Authors: Denef V. J. , Klappenbach J. A. , Patrauchan M. A. , Florizone C. , Rodrigues J. L. M. , Tsoi T. V. , Verstraete W. , Eltis L. D. , Tiedje J. M.
Published: January 2006

Transcriptomic and proteomic analyses of
Burkholderia xenovorans
LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol
ortho
cleavage (
ben
-
cat
) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (

box
C

) and the megaplasmid (

box
M

). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the
ben-cat
pathway (the Δ
benABCD
::
kan
mutant), the

box
C

pathway (the Δ

boxAB
C

::
kan
mutant), and both pathways (the Δ
benABCD
Δ

boxAB
C

::
kan
mutant). All three mutants oxidized benzoate in resting-cell assays. However, the Δ
benABCD
::
kan
and Δ
benABCD
Δ

boxAB
C

::
kan
mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the Δ
benABCD
::
kan
mutant growing on benzoate expressed both
box
pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of
benABCD
abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the
box
pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.
Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases
Authors: van Beilen Jan B. , Funhoff Enrico G. , van Loon Alexander , Just Andrea , Kaysser Leo , Bouza Manuel , Holtackers René , Röthlisberger Martina , Li Zhi , Witholt Bernard
Published: January 2006

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed
Pseudomonas putida
GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.
Benthic Bacterial and Fungal Productivity and Carbon Turnover in a Freshwater Marsh
Authors: Buesing Nanna , Gessner Mark O.
Published: January 2006

Heterotrophic bacteria and fungi are widely recognized as crucial mediators of carbon, nutrient, and energy flow in ecosystems, yet information on their total annual production in benthic habitats is lacking. To assess the significance of annual microbial production in a structurally complex system, we measured production rates of bacteria and fungi over an annual cycle in four aerobic habitats of a littoral freshwater marsh. Production rates of fungi in plant litter were substantial (0.2 to 2.4 mg C g
−1
C) but were clearly outweighed by those of bacteria (2.6 to 18.8 mg C g
−1
C) throughout the year. This indicates that bacteria represent the most actively growing microorganisms on marsh plant litter in submerged conditions, a finding that contrasts strikingly with results from both standing dead shoots of marsh plants and submerged plant litter decaying in streams. Concomitant measurements of microbial respiration (1.5 to 15.3 mg C-CO
2
g
−1
of plant litter C day
−1
) point to high microbial growth efficiencies on the plant litter, averaging 45.5%. The submerged plant litter layer together with the thin aerobic sediment layer underneath (average depth of 5 mm) contributed the bulk of microbial production per square meter of marsh surface (99%), whereas bacterial production in the marsh water column and epiphytic biofilms was negligible. The magnitude of the combined production in these compartments (∼1,490 g C m
−2
year
−1
) highlights the importance of carbon flows through microbial biomass, to the extent that even massive primary productivity of the marsh plants (603 g C m
−2
year
−1
) and subsidiary carbon sources (∼330 g C m
−2
year
−1
) were insufficient to meet the microbial carbon demand. These findings suggest that littoral freshwater marshes are genuine hot spots of aerobic microbial carbon transformations, which may act as net organic carbon importers from adjacent systems and, in turn, emit large amounts of CO
2
(here, ∼870 g C m
−2
year
−1
) into the atmosphere.
Prevalence and Transmission of Honeybee Viruses
Authors: Chen Y. P. , Pettis J. S. , Collins A. , Feldlaufer M. F.
Published: January 2006

Transmission mechanisms of six honeybee viruses, including acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood bee virus (SBV), in honey bee colonies were investigated by reverse transcription-PCR (RT-PCR) methods. The virus status of individual queens was evaluated by examining the presence of viruses in the queens' feces and tissues, including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body. Except for head tissue, all five tissues as well as queen feces were found to be positive for virus infections. When queens in bee colonies were identified as positive for BQCV, DWV, CBPV, KBV, and SBV, the same viruses were detected in their offspring, including eggs, larvae, and adult workers. On the other hand, when queens were found positive for only two viruses, BQCV and DWV, only these two viruses were detected in their offspring. The presence of viruses in the tissue of ovaries and the detection of the same viruses in queens' eggs and young larvae suggest vertical transmission of viruses from queens to offspring. To our knowledge, this is the first evidence of vertical transmission of viruses in honeybee colonies.
Slow-Release Inoculation Allows Sustained Biodegradation of γ-Hexachlorocyclohexane
Authors: Mertens Birgit , Boon Nico , Verstraete Willy
Published: January 2006

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (γ-hexachlorocyclohexane [γ-HCH]). Slow-release inoculation of
Sphingomonas
sp. γ 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the
linA
gene, encoding γ-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.
Responses of the Anaerobic Bacterial Community to Addition of Organic C in Chromium(VI)- and Iron(III)-Amended Microcosms
Authors: Kourtev Peter S. , Nakatsu Cindy H. , Konopka Allan
Published: January 2006

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO
2
) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the
Geobacteraceae
family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO
2
production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as
Pantoea
,
Pseudomonas
, and
Enterobacter
species.
Geobacteraceae
were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.
Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances
Authors: Sutka R. L. , Ostrom N. E. , Ostrom P. H. , Breznak J. A. , Gandhi H. , Pitt A. J. , Li F.
Published: January 2006

The intramolecular distribution of nitrogen isotopes in N
2
O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N
2
O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N
2
O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for
Nitrosomonas europaea
,
Nitrosospira multiformis
, and
Methylosinus trichosporium
, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N
2
O from ammonia oxidation by
N. europaea
(31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by
N. multiformis
(0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N
2
O produced during nitrite reduction by the denitrifiers
Pseudomonas chlororaphis
and
Pseudomonas aureofaciens
(−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ∼33‰ and ∼0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N
2
O.
Sources of
Campylobacter
spp. Colonizing Housed Broiler Flocks during Rearing
Authors: Bull S. A. , Allen V. M. , Domingue G. , Jørgensen F. , Frost J. A. , Ure R. , Whyte R. , Tinker D. , Corry J. E. L. , Gillard-King J. , Humphrey T. J.
Published: January 2006

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for
Campylobacter
spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and
flaA
restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period.
Campylobacter
spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house.
Campylobacter
spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.
Prevalence and Virulence Factors ofEscherichia coliSerogroups O26, O103, O111, and O145 Shed by Cattle in Scotland
Authors: Pearce M. C. , Evans J. , McKendrick I. J. , Smith A. W. , Knight H. I. , Mellor D. J. , Woolhouse M. E. J. , Gunn G. J. , Low J. C.
Published: January 2006

A national survey was conducted to determine the prevalence ofEscherichia coliO26, O103, O111, and O145 in feces of Scottish cattle. In total, 6,086 fecal pats from 338 farms were tested. The weighted mean percentages of farms on which shedding was detected were 23% forE. coliO26, 22% forE. coliO103, and 10% forE. coliO145. The weighted mean prevalences in fecal pats were 4.6% forE. coliO26, 2.7% forE. coliO103, and 0.7% forE. coliO145. NoE. coliO111 was detected. Farms with cattle sheddingE. coliserogroup O26, O103, or O145 were widely dispersed across Scotland and were identified most often in summer and autumn. However, on individual farms, fecal shedding ofE. coliO26, O103, or O145 was frequently undetectable or the numbers of pats testing positive were small. For serogroup O26 or O103 there was clustering of positive pats within management groups, and the presence of an animal shedding one of these serogroups was a positive predictor for shedding by others, suggesting local transmission of infection. Carriage ofvtxwas rare inE. coliO103 and O145 isolates, but 49.0% ofE. coliO26 isolates possessedvtx, invariablyvtx1alone orvtx1andvtx2together. The carriage ofeaeandehxAgenes was highly associated in all three serogroups. AmongE. coliserogroup O26 isolates, 28.9% carriedvtx,eae, andehxA—a profile consistent withE. coliO26 strains known to cause human disease.
Quantification of
Campylobacter
Species Cross-Contamination during Handling of Contaminated Fresh Chicken Parts in Kitchens
Authors: Luber Petra , Brynestad Sigrid , Topsch Daniela , Scherer Kathrin , Bartelt Edda
Published: January 2006

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling
Campylobacter
-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of
Campylobacter
cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of
Campylobacter
and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the
Campylobacter
transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of
Campylobacter
present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (
P
< 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.
Protective Effects of Organic Acids on Survival of
Escherichia coli
O157:H7 in Acidic Environments
Authors: Bjornsdottir K. , Breidt F. , McFeeters R. F.
Published: January 2006

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of
Escherichia coli
O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted
E. coli
O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for
E. coli
O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or
l
-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the
E. coli
cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the
E. coli
cells.
d
-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of
E. coli
O15:H7 under low-pH conditions.
Leaching of Phage from Class B Biosolids and Potential Transport through Soil
Authors: Chetochine Alexandra S. , Brusseau Mark L. , Gerba Charles P. , Pepper Ian L.
Published: January 2006

The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to
Escherichia coli
ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.
Impact of Inoculum Preparation and Storage Conditions on the Response of
Escherichia coli
O157:H7 Populations to Undercooking and Simulated Exposure to Gastric Fluid
Authors: Stopforth Jarret D. , Skandamis Panagiotis N. , Ashton Laura V. , Geornaras Ifigenia , Kendall Patricia A. , Belk Keith E. , Scanga John A. , Smith Gary C. , Sofos John N.
Published: January 2006

This study evaluated the impact of inoculum preparation and storage conditions on the response of
Escherichia coli
O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with
E. coli
O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75°C) water followed by lactic acid (2%, 55°C), vacuum packaged, stored at 4 (28 days) or 12°C (16 days), and periodically transferred to aerobic storage (7°C for 5 days). During storage, samples were exposed to sequential heat (55°C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4°C was lower than that of cells on nondecontaminated meat stored at 12°C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12°C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4°C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12°C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.
Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of
Aspergillus carbonarius
,
Aspergillus tubingensis
, and
Aspergillus niger
Strains Isolated from Grapes in Italy
Authors: Perrone Giancarlo , Mulè Giuseppina , Susca Antonia , Battilani Paola , Pietri Amedeo , Logrieco Antonio
Published: January 2006

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than
Aspergillus carbonarius
within
Aspergillus
section
Nigri
to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of
A. carbonarius
produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of
A. tubingensis
produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of
A. niger
produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of
Aspergillus
“uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that
A. tubingensis
is able to produce ochratoxin and that, together with
A. carbonarius
and
A. niger
, it may be responsible for the ochratoxin contamination of wine in Italy.
Anaerobic Nitrate-Dependent Iron(II) Bio-Oxidation by a Novel Lithoautotrophic Betaproteobacterium, Strain 2002
Authors: Weber Karrie A. , Pollock Jarrod , Cole Kimberly A. , O'Connor Susan M. , Achenbach Laurie A. , Coates John D.
Published: January 2006

Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 × 10
3
to 1.5 × 10
4
cells g
−1
wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to
Chromobacterium violaceum
, a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and
C. violaceum
incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N
2
and N
2
O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO
2
for growth as well as the assimilation of
14
C-labeled CO
2
into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.
Diverse AvrPtoB Homologs from SeveralPseudomonas syringaePathovars Elicit Pto-Dependent Resistance and Have Similar Virulence Activities
Authors: Lin Nai-Chun , Abramovitch Robert B. , Kim Young Jin , Martin Gregory B.
Published: January 2006

AvrPtoB is a type III effector protein fromPseudomonas syringaepv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously thatavrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization ofavrPtoBhomologs fromP. syringaepv. tomato T1, PT23, and JL1065,P. syringaepv. syringae B728a, andP. syringaepv. maculicola ES4326. TheavrPtoBhomolog fromP. syringaepv. maculicola,hopPmaL, was identified previously. The four new genes identified in this study are designatedavrPtoBT1,avrPtoBPT23,avrPtoBJL1065, andavrPtoBB728a. The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of theavrPtoBhomologs were detected in thePseudomonasstrains from which they were isolated. Proteins encoded by the homologs were detected in all strains exceptP. syringaepv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoBT1. All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited aPto-dependent defense response when they were delivered into leaf cells by DC3000ΔavrPtoΔavrPtoB, aP. syringaepv. tomato strain with a deletion of bothavrPtoandavrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000ΔavrPtoΔavrPtoBto form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in anAgrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverseP. syringaepathovars have conserved avirulence and virulence activities similar to AvrPtoB activity.
Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in
Bacillus subtilis
168
Authors: Kurosawa Kazuhiko , Hosaka Takeshi , Tamehiro Norimasa , Inaoka Takashi , Ochi Kozo
Published: January 2006

The capacity of ribosomal modification to improve antibiotic production by
Streptomyces
spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (
str
) also enhances α-amylase (and protease) production by a strain of
Bacillus subtilis
as estimated by measuring the enzyme activity. The
str
mutations are point mutations within
rpsL
, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various
rpsL
mutations K56R (which corresponds to position 42 in
E. coli
) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg
2+
concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.
Recurring Seasonal Dynamics of Microbial Communities in Stream Habitats
Authors: Hullar Meredith A. J. , Kaplan Louis A. , Stahl David A.
Published: January 2006

Recurring seasonal patterns of microbial distribution and abundance in three third-order temperate streams within the southeast Pennsylvania Piedmont were observed over 4 years. Populations associated with streambed sediments and rocks (epilithon) were identified using terminal restriction length polymorphism (tRFLP) and sequencing of 16S rRNA genes selectively amplified with primers for the bacterial domain. Analyses of the relative magnitudes of tRFLP peak areas by using nonmetric multidimensional scaling resolved clear seasonal trends in epilithic and sediment populations. Oscillations between two dominant groups of epilithic genotypes, explaining 86% of the seasonal variation in the data set, were correlated with temperature and dissolved organic carbon. Sequences affiliated with epilithic phototrophs (cyanobacteria and diatom chloroplasts), a
Rhodoferax
sp., and a
Bacillus
species clustered in the summer, whereas sequences most closely related to “
Betaproteobacteria
” (putative
Burkholderia
sp.), and a putative cyanobacterium clustered in the fall/spring. The sediment genotypes also clustered into two groups, and these explained 85% of seasonal variation but correlated only with temperature. A summer tRFLP pattern was characterized by prevalence of “
Betaproteobacteria
,” “
Gammaproteobacteria
,” and a
Bacillus
sp., whereas the winter/spring pattern was characterized by phylotypes most closely related to “
Firmicutes
,” “
Gammaproteobacteria
,” and “
Nitrospirae
.” A close association between these headwater streams and their watersheds was suggested by the recovery of sequences related to microbial populations provisionally attributed to not only freshwaters but also terrestrial habitats.
Prochlorococcus
Ecotype Abundances in the North Atlantic Ocean As Revealed by an Improved Quantitative PCR Method
Authors: Zinser Erik R. , Coe Allison , Johnson Zackary I. , Martiny Adam C. , Fuller Nicholas J. , Scanlan David J. , Chisholm Sallie W.
Published: January 2006

The cyanobacterium
Prochlorococcus
numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of
Prochlorococcus
have been described, and their distinctive physiologies and genomes indicate that these lineages are “ecotypes” and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total
Prochlorococcus
counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.
Making All Parts of the 16S rRNA of
Escherichia coli
Accessible In Situ to Single DNA Oligonucleotides
Authors: Yilmaz L. Şafak , Ökten Hatice E. , Noguera Daniel R.
Published: January 2006

rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of
Escherichia coli
to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (Δ
G
o
overall
) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of
E. coli
could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving Δ
G
o
overall
, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.
Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by RecombinantLactobacillus caseiCells
Authors: Aires Karina Araujo , Cianciarullo Aurora Marques , Carneiro Sylvia Mendes , Villa Luisa Lina , Boccardo Enrique , Pérez-Martinez Gaspar , Perez-Arellano Isabel , Oliveira Maria Leonor Sarno , Ho Paulo Lee
Published: January 2006

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on theLactobacillus caseilactose operon promoter (plac) for expression of the HPV-16 L1 protein inL. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis ofL. caseiexpressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on theL. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized withL. caseiexpressing L1 reacted withSpodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs byLactobacillusopens the possibility for development of new live mucosal prophylactic vaccines.
Multicopy Integration and Expression of Heterologous Genes in
Methylobacterium extorquens
ATCC 55366
Authors: Choi Young J. , Bourque Denis , Morel Lyne , Groleau Denis , Míguez Carlos B.
Published: January 2006

High-level expression of chromosomally integrated genes in
Methylobacterium extorquens
ATCC 55366 was achieved under the control of the strong
M. extorquens
AM1 methanol dehydrogenase promoter (P

mxaF

) using the mini-Tn
7
transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for
M. extorquens
was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (
gfp
) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn
7
integration locus in the chromosome of
M. extorquens
, known as the Tn
7
attachment site (
att
Tn
7
site), was identified. This single
att
Tn
7
site was identified in an intergenic region between
glmS
, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and
dhaT
, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the
att
Tn
7
site is a noncoding region of the chromosome make the mini-Tn
7
transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (
bgl
[β-galactosidase],
est
[esterase], and
gfp
[green fluorescent protein]) in
M. extorquens
.
Competitive Fitness of
Listeria monocytogenes
Serotype 1/2a and 4b Strains in Mixed Cultures with and without Food in the U.S. Food and Drug Administration Enrichment Protocol
Authors: Gorski Lisa , Flaherty Denise , Mandrell Robert E.
Published: January 2006

Thirteen different serotypes of the food-borne pathogen
Listeria monocytogenes
have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence
L. monocytogenes
is described in the U.S. Food and Drug Administration (FDA)
Bacterial Analytical Manual
(BAM). We compared three strains of
L. monocytogenes
serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate
Listeria
species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.