bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization, Seasonal Occurrence, and Diel Fluctuation of Poly(hydroxyalkanoate) in Photosynthetic Microbial Mats
Authors: Rothermich Mary M. , Guerrero Ricardo , Lenz Robert W. , Goodwin Steve 
Published: July 2002

ABSTRACTIn situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1% of the organic carbon in the green layer and up to 6% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.
Marine Planktonic Archaea Take Up Amino Acids
Authors: Ouverney Cleber C. , Fuhrman Jed A. 
Published: July 2002

ABSTRACT
Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.
Relationships between Colony Morphotypes and Oil Tolerance in
Rhodococcus rhodochrous
Authors: Iwabuchi Noriyuki , Sunairi Michio , Anzai Hirosi , Nakajima Mutsuyasu , Harayama Shigeaki 
Published: July 2002

ABSTRACT

A mucoidal strain of
Rhodococcus rhodochrous
was resistant to 10% (vol/vol)
n
-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance to
n
-hexadecane. Thus, EPS confer tolerance to
n
-hexadecane in members of the genus
Rhodococcus.
Enrichment and Molecular Characterization of a Bacterial Culture That Degrades Methoxy-Methyl Urea Herbicides and Their Aniline Derivatives
Authors: El-Fantroussi Said 
Published: July 2002

ABSTRACTSoil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3,4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37°C, 3,4-dichloroaniline was transformed only at 28°C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates.
Leaching of
Escherichia coli
O157:H7 in Diverse Soils under Various Agricultural Management Practices
Authors: Gagliardi Joel V. , Karns Jeffrey S. 
Published: July 2002

ABSTRACT

Application of animal manures to soil as crop fertilizers is an important means for recycling the nitrogen and phosphorus which the manures contain. Animal manures also contain bacteria, including many types of pathogens. Manure pathogen levels depend on the source animal, the animal's state of health, and how the manure was stored or treated before use. Rainfall may result in pathogen spread into soil by runoff from stored or unincorporated manure or by leaching through the soil profile. Steady rainfall consisting of 16.5 mm h
−1
was applied to 100-mm disturbed soil cores that were treated with manure and inoculated with
Escherichia coli
O157:H7 strain B6914. The level of B6914 in leachate was near the inoculum level each hour for 8 h, as was the level of B6914 at several soil depths after 24 h, indicating that there was a high rate of growth. Bacterial movement through three different types of soil was then compared by using disturbed (tilled) and intact (no-till) soil cores and less intense rainfall consisting of 25.4 mm on 4 consecutive days and then four more times over a 17-day period. Total B6914 levels exceeded the inoculum levels for all treatments except intact clay loam cores. B6914 levels in daily leachate samples decreased sharply with time, although the levels were more constant when intact sandy loam cores were used. The presence of manure often increased total B6914 leachate and soil levels in intact cores but had the opposite effect on disturbed soil cores. Ammonia and nitrate levels correlated with B6914 and total coliform levels in leachate. We concluded that tillage practice, soil type, and method of pathogen delivery affect but do not prevent vertical
E. coli
O157:H7 and coliform transport in soil and that soluble nitrogen may enhance transport.
Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth
Authors: Picard C. , Di Cello F. , Ventura M. , Fani R. , Guckert A. 
Published: July 2002

ABSTRACTAPseudomonas2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil ofZea maysand in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing theAluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.
Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of
Lactococcus lactis
Authors: Fernández Leonides , Beerthuyzen Marke M. , Brown Julie , Siezen Roland J. , Coolbear Tim , Holland Ross , Kuipers Oscar P. 
Published: July 2002

ABSTRACT

The gene encoding the major intracellular tributyrin esterase of
Lactococcus lactis
was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named
estA
, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the
estA
product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in
L. lactis
by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The
estA
gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type
estA
gene. This confirms that EstA is the main enzyme responsible for esterase activity in
L. lactis
. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to
S
-formylglutathione hydrolase (FGH) of
Paracoccus denitrificans
and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.
Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids
Authors: Hohnstock A. M. , Stuart-Keil K. G. , Kull E. E. , Madsen E. L. 
Published: July 2002

ABSTRACT
We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.
Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant
Escherichia coli
Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics
Authors: Kansiz Mustafa , Billman-Jacobe Helen , McNaughton Don 
Published: July 2002

ABSTRACT
Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(β-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.
Simultaneous Recovery of RNA and DNA from Soils and Sediments
Authors: Hurt Richard A. , Qiu Xiaoyun , Wu Liyou , Roh Yul , Palumbo A. V. , Tiedje J. M. , Zhou Jizhong 
Published: July 2002

ABSTRACTRecovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316–322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0°C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 μg g of soil−1dry weight), whereas the DNA yield ranged from 23 to 435 μg g−1. In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.
Clostridium beijerinckii
Cells Expressing
Neocallimastix patriciarum
Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production
Authors: López-Contreras Ana M. , Smidt Hauke , van der Oost John , Claassen Pieternel A. M. , Mooibroek Hans , de Vos Willem M. 
Published: July 2002

ABSTRACT

Growth and the production of acetone, butanol, and ethanol by
Clostridium beijerinckii
NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases,
celA
and
celD
from the fungus
Neocallimastix patriciarum
, were cloned separately into
C. beijerinckii
. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the
Clostridium saccharobutylicum
NCP262
eglA
gene was constructed and fused either to the
celA
gene or the
celD
gene. Stable
C. beijerinckii
transformants were obtained with the resulting plasmids, pWUR3 (
celA
) and pWUR4 (
celD
). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring
celA
and 78 U/mg of protein for transformants harboring
celD
). Although
C. beijerinckii
harboring either
celA
or
celD
was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.
Effects of Combined Water Potential and Temperature Stresses on
Cryptosporidium parvum
Oocysts
Authors: Walker Mark , Leddy Katherine , Hagar Elaine 
Published: July 2002

ABSTRACT

Hosts infected with the parasite
Cryptosporidium parvum
may excrete oocysts on soils in watersheds that supply public drinking water. Environmental stresses decrease the numbers of oocysts after deposition on soils. However, the rates and effects of combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to combined stresses of water potential (−4, −12, and −33 bars), above-freezing temperatures (4 and 30°C), and a subfreezing temperature (−14°C) for 1, 14, and 29 days and one to six freeze-thaw cycles (−14 to 10°C) to estimate coefficients to characterize population degradation using multiplicative error and exponential decay models. The experiments were carried out in NaCl solutions with water potentials of −4, −12, and −33 bars, in combination with temperature stresses at levels that could be expected in natural soils. Increased water potential increased the rate of population degradation for all temperature conditions investigated. Enhanced degradation leads to estimated rates of population degradation that are greater than those that have been reported and used in previous studies conducted to assess risk of water supply contamination from sources of
C. parvum
.
Multiplex PCR Assay for Detection and Identification ofClostridium botulinumTypes A, B, E, and F in Food and Fecal Material
Authors: Lindström Miia , Keto Riikka , Markkula Annukka , Nevas Mari , Hielm Sebastian , Korkeala Hannu 
Published: July 2002

ABSTRACTBotulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinumcells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection ofClostridium botulinumtypes A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43C. botulinumstrains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp forC. botulinumtype A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, includingC. sporogenesand the nontoxigenic nonproteolyticC. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2spore/g for types A, B, and F to 10−1spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to containC. botulinumtype A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics ofC. botulinum.
Effects of Nondigestible Oligosaccharides onSalmonella entericaSerovar Typhimurium and NonpathogenicEscherichia coliin the Pig Small Intestine In Vitro
Authors: Naughton Patrick J. , Mikkelsen Lene Lind , Jensen Bent Borg 
Published: July 2002

ABSTRACTAn in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with eitherSalmonellaor nonpathogenicEscherichia colistrains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenicE. coliand ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease inE. coliassociated with the jejunum in the presence of mannose (P< 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers ofE. coliin jejunal organ cultures of pigs fed the FOS diet (P< 0.05). There was a reduction, though not a significant one, in the association ofSalmonellasp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affectE. coliorSalmonellanumbers.
Fate ofMycobacterium aviumsubsp.paratuberculosisin Swiss Hard and Semihard Cheese Manufactured from Raw Milk
Authors: Spahr U. , Schafroth K. 
Published: July 2002

ABSTRACTRaw milk was artificially contaminated with declumped cells ofMycobacterium aviumsubsp.paratuberculosisat a concentration of 104to 105CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains ofM. aviumsubsp.paratuberculosiswere tested, and for each strain, two model hard and semihard cheeses were produced. The survival ofM. aviumsubsp.paratuberculosiscells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese.Dvalues were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death ofM. aviumsubsp.paratuberculosisin cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, theDvalues found indicate that 103to 104cells ofM. aviumsubsp.paratuberculosisper g will be inactivated.