bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Microbial Community Composition and Function during a Polyaromatic Hydrocarbon Phytoremediation Field Trial
Authors: Siciliano Steven D. , Germida James J. , Banks Kathy , Greer Charles W.
Published: January 2003

The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil. The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed. In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (
ndoB
,
alkB
, and
xylE)
as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants. There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence. Tall Fescue (
Festuca arundinacea)
increased the prevalence of
ndoB
,
alkB
, and
xylE
as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil. In contrast, Rose Clover (
Trifolium hirtum)
decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil. The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community. This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation.
Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products
Authors: Courtois Sophie , Cappellano Carmela M. , Ball Maria , Francou Francois-Xavier , Normand Philippe , Helynck Gérard , Martinez Asuncion , Kolvek Steven J. , Hopke Joern , Osburne Marcia S. , August Paul R. , Nalin Renaud , Guérineau Michel , Jeannin Pascale , Simonet Pascal , Pernodet Jean-Luc
Published: January 2003

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone “shotgun” environmental DNA library by using an
Escherichia coli-Streptomyces lividans
shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.
Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing
Arabidopsis thaliana
Phytochelatin Synthase
Authors: Sauge-Merle Sandrine , Cuiné Stéphan , Carrier Patrick , Lecomte-Pradines Catherine , Luu Doan-Trung , Peltier Gilles
Published: January 2003

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the
Arabidopsis thaliana
gene encoding PC synthase (
AtPCS
) was expressed in
Escherichia coli.
A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.
Optimal Growth and Ethanol Production from Xylose by Recombinant
Saccharomyces cerevisiae
Require Moderate
d
-Xylulokinase Activity
Authors: Jin Yong-Su , Ni Haiying , Laplaza Jose M. , Jeffries Thomas W.
Published: January 2003

d
-Xylulokinase (XK) is essential for the metabolism of
d
-xylose in yeasts. However, overexpression of genes for XK, such as the
Pichia stipitis XYL3
gene and the
Saccharomyces cerevisiae XKS
gene, can inhibit growth of
S
.
cerevisiae
on xylose. We varied the copy number and promoter strength of
XYL3
or
XKS1
to see how XK activity can affect xylose metabolism in
S
.
cerevisiae
. The
S
.
cerevisiae
genetic background included single integrated copies of
P
.
stipitis XYL1
and
XYL2
driven by the
S
.
cerevisiae TDH1
promoter. Multicopy and single-copy constructs with either
XYL3
or
XKS1
, likewise under control of the
TDH1
promoter, or with the native
P
.
stipitis
promoter were introduced into the recombinant
S
.
cerevisiae
. In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of
XYL3
and
XKS1
inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant
S
.
cerevisiae
is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an
S
.
cerevisiae tps1
mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing
XYL3
under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of
XYL3
per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.
Gene Cloning, Purification, and Characterization of a Phosphodiesterase from
Delftia acidovorans
Authors: Tehara Sundiep K. , Keasling Jay D.
Published: January 2003

A novel phosphodiesterase (PdeA) was purified from
Delftia acidovorans
, the gene encoding the enzyme was cloned and expressed in
Escherichia coli
, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(
p
-nitrophenyl)phosphate with a

K
m

of 2.9 ± 0.1 mM and a
k
cat
of 879 ± 73 min
−1
. The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in
E. coli
. The IS
1071
transposon insertion sequence was found downstream of
pdeA
.
Genetic Variability within
Borrelia burgdorferi
Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the
rrfA-rrlB
Intergenic Spacer in
Ixodes ricinus
Ticks from the Czech Republic
Authors: Derdáková Markéta , Beati Lorenza , Pet'ko Branislav , Stanko Michal , Fish Durland
Published: January 2003

In Europe the
Borrelia burgdorferi
sensu lato complex is represented by five distinct genospecies:
Borrelia burgdorferi
sensu stricto,
Borrelia afzelii
,
Borrelia garinii
,
Borrelia valaisiana
, and
Borrelia lusitaniae.
These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish
Borrelia
strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370
Ixodes ricinus
ticks collected in the Czech Republic was analyzed by PCR for the presence of a ∼230-bp fragment of the
rrfA-rrlB
intergenic spacer of
Borrelia
spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme
Mse
I and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown
Borrelia
genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.
Gene Replacement in Mycobacteria by Using Incompatible Plasmids
Authors: Pashley Carey A. , Parish Tanya , McAdam Ruth A. , Duncan Ken , Stoker Neil G.
Published: January 2003

A simple and efficient delivery system was developed for making targeted gene knockouts in
Mycobacterium smegmatis
. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (
repA
and
repB
), which act in
trans
, and the use of vectors that each lack one
rep
gene and complement each other resulted in the loss of both plasmids in
M. smegmatis
and
Mycobacterium bovis
BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that
M. smegmatis pyrF
mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including
Mycobacterium tuberculosis
.
Genes for Chlorogenate and Hydroxycinnamate Catabolism (
hca
) Are Linked to Functionally Related Genes in the
dca-pca-qui-pob-hca
Chromosomal Cluster of
Acinetobacter
sp. Strain ADP1
Authors: Smith Michael A. , Weaver Valerie B. , Young David M. , Ornston L. Nicholas
Published: January 2003

Hydroxycinnamates are ubiquitous in the environment because of their contributions to the structure and defense mechanisms of plants. Additional plant products, many of which are formed in response to stress, support the growth of
Acinetobacter
sp. strain ADP1 through pathways encoded by genes in the
dca
-
pca-qui
-
pob
chromosomal cluster. In an appropriate genetic background, it was possible to select for an
Acinetobacter
strain that had lost the ability to grow with caffeate, a commonly occurring hydroxycinnamate. The newly identified mutation was shown to be a deletion in a gene designated
hcaC
and encoding a ligase required for conversion of commonly occurring hydroxycinnamates (caffeate, ferulate, coumarate, and 3,4-dihydroxyphenylpropionate) to thioesters. Linkage analysis showed that
hcaC
is linked to
pobA
. Downstream from
hcaC
and transcribed in the direction opposite the direction of
pobA
transcription are open reading frames designated
hcaDEFG
. Functions of these genes were inferred from sequence comparisons and from the properties of knockout mutants. HcaD corresponded to an acyl coenzyme A (acyl-CoA) dehydrogenase required for conversion of 3,4-dihydroxyphenylpropionyl-CoA to caffeoyl-CoA. HcaE appears to encode a member of a family of outer membrane proteins known as porins. Knockout mutations in
hcaF
confer no discernible phenotype. Knockout mutations in
hcaG
indicate that this gene encodes a membrane-associated esterase that hydrolyzes chlorogenate to quinate, which is metabolized in the periplasm, and caffeate, which is metabolized by intracellular enzymes. The chromosomal location of
hcaG
, between
hcaC
(required for growth with caffeate) and
quiA
(required for growth with quinate), provided the essential clue that led to the genetic test of HcaG as the esterase that produces caffeate and quinate from chlorogenate. Thus, in this study, organization within what is now established as the
dca
-
pca-qui
-
pob
-
hca
chromosomal cluster provided essential information about the function of genes in the environment.
Molecular Characterization ofLegionellaPopulations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops
Authors: Calvo-Bado Leo A. , Morgan J. Alun W. , Sergeant Martin , Pettitt Tim R. , Whipps John M.
Published: January 2003

The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology toLegionellaspecies, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by usingLegionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a singleLegionellastrain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that severalLegionellaspecies were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number ofLegionellacells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth ofLegionellaand should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.
Diversity, Frequency, and Persistence ofEscherichia coliO157 Strains from Range Cattle Environments
Authors: Renter David G. , Sargeant Jan M. , Oberst Richard D. , Samadpour Mansour
Published: January 2003

Genetic diversity, isolation frequency, and persistence were determined forEscherichia coliO157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively consideredE. coliO157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to beE. coliO157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) ofXbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to beE. coliO157. By analyzing up to three isolates per positive sample, we detected an average of 1.80XbaI-PFGE subtypes per sample. MostXbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of theE. coliO157 isolates (124 of 235 isolates). RecurringXbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. SevenXbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphissp.). The number ofXbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology ofE. coliO157 previously described for confined cattle operations is also evident in extensively managed range cattle environments.
Growth Dynamics ofSalmonella entericaStrains on Alfalfa Sprouts and in Waste Seed Irrigation Water
Authors: Howard Michael B. , Hutcheson Steven W.
Published: January 2003

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds bySalmonellabacteria that developed into clinically significant populations during the seed germination process. To test the possibility thatSalmonella entericastrains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. AnS. entericaserovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication ofS. entericacells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability ofS. entericato grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. TheS. entericastrains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source.S. entericastrains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detectS. entericain the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.
Longitudinal Study ofEscherichia coliO157 in a Cattle Finishing Unit
Authors: Lahti Elina , Ruoho Olli , Rantala Leila , Hänninen Marja-Liisa , Honkanen-Buzalski Tuula
Published: January 2003

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources ofEscherichia coliO157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative forE. coliO157 on arrival, whereas positive animals were detected 1 day later. During the fattening period theE. coliO157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months.E. coliO157 was isolated from samples taken from water cups, floors, and feed passages.E. coliO157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive.E. coliO157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P< 0.0001). Fecal samples taken at the abattoir had lower counts (≤0.4 MPN/g) than fecal samples at the farm (P< 0.05).E. coliO157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P< 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source ofE. coliO157 at the farm and thatE. coliO157 seemed to persist well on barn surfaces.
ACEI of
Trichoderma reesei
Is a Repressor of Cellulase and Xylanase Expression
Authors: Aro Nina , Ilmén Marja , Saloheimo Anu , Penttilä Merja
Published: January 2003

We characterized the effect of deletion of the
Trichoderma reesei
(
Hypocrea jecorina
)
ace1
gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in
Saccharomyces cerevisiae
the promoter of the main cellulase gene,
cbh1
. Deletion of
ace1
resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the
ace1
gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δ
ace1
strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the
ace1
gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the
ace1
gene and with a deletion of the
ace2
gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δ
ace1
strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.
Acetic Acid Increases Stability of Silage under Aerobic Conditions
Authors: Danner H. , Holzer M. , Mayrhuber E. , Braun R.
Published: January 2003

The effects of various compounds on the aerobic stability of silages were evaluated. It has been observed that inoculation of whole-crop maize with homofermentative lactic acid bacteria leads to silages which have low stability against aerobic deterioration, while inoculation with heterofermentative lactic acid bacteria, such asLactobacillus brevisorLactobacillus buchneri, increases stability. Acetic acid has been proven to be the sole substance responsible for the increased aerobic stability, and this acid acts as an inhibitor of spoilage organisms. Therefore, stability increases exponentially with acetic acid concentration. Only butyric acid has a similar effect. Other compounds, like lactic acid, 1,2-propanediol, and 1-propanol, have been shown to have no effect, while fructose and mannitol reduce stability.
2-
n
-Pentyl-4-Quinolinol Produced by a Marine
Alteromonas
sp. and Its Potential Ecological and Biogeochemical Roles
Authors: Long Richard A. , Qureshi Asfia , Faulkner D. John , Azam Farooq
Published: January 2003

Bacterium-bacterium interactions occur at intimate spatial scales on the order of micrometers, but our knowledge of interactions at this level is rudimentary. Antagonism is a potential interaction in such microenvironments. To study the ecological role of antibiosis, we developed a model system involving an antibiotic-producing isolate (SWAT5) derived from a marine particle and its dominant antibiotic product, 2-
n
-pentyl-4-quinolinol (PQ). This system was used to address questions about the significance of this antibiotic for microbial ecology and carbon cycling on particles. We characterized the chemical and inhibitory properties of PQ in relation to the mechanisms used by particle-associated bacteria in interacting with particles and with other attached bacteria. PQ was produced by SWAT5 only on surfaces. When SWAT5 was grown in polysaccharide matrices, PQ diffused within the matrices but not into the surrounding seawater. SWAT5 might thus be able to generate a localized zone of high antibiotic concentration on particles suspended or sinking through seawater. Target bacterial respiration was most sensitive to PQ (75 nM), while inhibition of DNA synthesis, protein synthesis, and bacterial motility required higher (micromolar) PQ levels. The presence of PQ altered the composition of the bacterial community that colonized and developed in a model particle system. PQ also inhibited
Synechococcus
and phytoplankton growth. Our results suggest that antibiosis may significantly influence community composition and activities of attached bacterial and thus regulate the biogeochemical fate of particulate organic matter in the ocean.
Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation
Authors: Thurston-Enriquez Jeanette A. , Haas Charles N. , Jacangelo Joseph , Riley Kelley , Gerba Charles P.
Published: January 2003

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm
2
, respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm
2
in treated groundwater. A dose of 103 mJ/cm
2
was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.
Influences of Pond Geochemistry, Temperature, and Freeze-Thaw on Terminal Anaerobic Processes Occurring in Sediments of Six Ponds of the McMurdo Ice Shelf, near Bratina Island, Antarctica
Authors: Mountfort Douglas O. , Kaspar Heinrich F. , Asher Rodney A. , Sutherland Donna
Published: January 2003

The effects of freeze-thaw, freezing and sediment geochemistry on terminal anaerobic processes occurring in sediments taken from below cyanobacterial mats in meltwater ponds of the McMurdo Ice Shelf in Antarctica were investigated. Depending on the geochemical and physical status of the sediments (i.e., frozen or thawed), as well as passage of sediment through a freeze-thaw cycle, terminal carbon and electron flow shifted in which the proportions of hydrogen and acetate utilized for methanogenesis and sulfate reduction changed. Thus, in low-sulfate (or chloride) sediment which was thawed and incubated at 4°C, total carbon and electron flow were mediated by acetate-driven sulfate reduction and H
2
-driven methanogenesis. When the same sediments were incubated frozen, both methanogenesis and sulfate reduction decreased. However, under these conditions methanogenesis was favored over sulfate reduction, and carbon flow from acetate to methane increased relative to sulfate reduction; >70% of methane was contributed by acetate, and more than 80% of acetate was oxidized by pathways not coupled to sulfate reduction. In high-sulfate pond sediments, sulfate reduction was a major process mediating terminal carbon and electron flow in both unfrozen and frozen incubations. However, as with low-sulfate sediments, acetate oxidation became uncoupled from sulfate reduction with freezing. Geochemical and temperature effects could be expressed by linear models in which the log (methanogenesis to sulfate reduction) was negative log linear with respect to either temperature or the log of the sulfate (or chloride) concentration. From these relationships it was possible to predict the ratio for a given temperature (low-sulfate sediments) or sulfate (chloride) concentration. Small transitory changes, such as elevated sulfate reduction coupled to increased acetate turnover, resulted from application of a freeze-thaw cycle to low-salinity pond sediments. The results demonstrate how ecophysiological processes may change in anaerobic systems under extreme conditions (e.g., freezing) and provide new insights into microbial events occurring under these conditions.
A Field Investigation of
Bacillus anthracis
Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001
Authors: Higgins James A. , Cooper Mary , Schroeder-Tucker Linda , Black Scott , Miller David , Karns Jeffrey S. , Manthey Erlynn , Breeze Roger , Perdue Michael L.
Published: January 2003

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of
Bacillus anthracis
spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for
B. anthracis
at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected
B. anthracis
colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for
capA
and
vrr
genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of
B. anthracis
, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.
Survival and Growth ofFrancisella tularensisinAcanthamoeba castellanii
Authors: Abd Hadi , Johansson Thorsten , Golovliov Igor , Sandström Gunnar , Forsman Mats
Published: January 2003

Francisella tularensisis a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis,Acanthamoeba castellaniiwas cocultured with a strain ofF. tularensisengineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence withinA. castellaniiwas then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location ofF. tularensisinA. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that manyF. tularensiscells were located intracellularly inA. castellaniicells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells.F. tularensiswas found in intact trophozoites, excreted vesicles, and cysts. Furthermore,F. tularensisgrew faster in cocultures withA. castellaniithan it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number ofA. castellaniicells. The interaction betweenF. tularensisand amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir forF. tularensis.
Bacteria Associated with Cysts of the Soybean Cyst Nematode (
Heterodera glycines
)
Authors: Nour Sarah M. , Lawrence John R. , Zhu Hong , Swerhone George D. W. , Welsh Martha , Welacky Tom W. , Topp Edward
Published: January 2003

The soybean cyst nematode (SCN),
Heterodera glycines
, causes economically significant damage to soybeans (
Glycine max
) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) × 10
5
bacteria (mean ± standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera
Actinomadura
and
Streptomyces
. A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by
Lysobacter
and
Variovorax
spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.
Genetic Diversity of Isolates of
Glomus mosseae
from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis
Authors: Giovannetti Manuela , Sbrana Cristiana , Strani Patrizia , Agnolucci Monica , Rinaudo Valeria , Avio Luciano
Published: January 2003

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species
Glomus mosseae
. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of
G. mosseae
revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.
Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut
Authors: Noda Satoko , Ohkuma Moriya , Yamada Akinori , Hongoh Yuichi , Kudo Toshiaki
Published: January 2003

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite
Neotermes koshunensis
were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus
Treponema
and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite
Treponema
cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of
N. koshunensis
. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species,
Reticulitermes speratus
and
Hodotermopsis sjoestedti
, failed to detect any spirochete population. The absence of cluster II spirochetes in
N. koshunensis
was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species
Devescovina
sp.,
Stephanonympha
sp., and
Oxymonas
sp. in
N. koshunensis
were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.
Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products
Authors: Lavermicocca Paola , Valerio Francesca , Visconti Angelo
Published: January 2003

Phenyllactic acid (PLA) has recently been found in cultures of
Lactobacillus plantarum
that show antifungal activity in sourdough breads. The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of
Aspergillus
,
Penicillium
, and
Fusarium
that were isolated from bakery products, flours, or cereals. Less than 7.5 mg of PLA ml
−1
was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of ≤10 mg ml
−1
. Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml
−1
in buffered medium at pH 4, which is a condition more similar to those in real food systems. Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of
Penicillium verrucosum
and
Penicillium citrinum
and a strain of
Penicillium roqueforti
(the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains. The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated. The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs.
Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization
Authors: Nielsen Jeppe Lund , Aquino de Muro Marilena , Nielsen Per Halkjær
Published: January 2003

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in
Thiothrix
filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active
Thiothrix
cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.
Distribution of Microorganisms in the Subsurface of the Manus Basin Hydrothermal Vent Field in Papua New Guinea
Authors: Kimura Hiroyuki , Asada Ryuji , Masta Andrew , Naganuma Takeshi
Published: January 2003

The distribution of microorganisms in the subsurfaces of hydrothermal vents was investigated by using subvent rock core samples. Microbial cells and ATP were detected from cores taken at depths of less than 99.4 and 44.8 m below the seafloor (mbsf), respectively. Cores from various depths were incubated anaerobically with a heterotrophic medium. Growth at 60 and 90°C was ascribed to a
Geobacillus
sp. in the 448.6- to 99.4-mbsf cores and a
Deinococcus
sp. in the 64.8- to 128.9-mbsf cores, respectively, based on the 16S ribosomal DNA analysis.
The Spoilage Yeast
Zygosaccharomyces bailii
Forms Mitotic Spores: a Screening Method for Haploidization
Authors: Rodrigues Fernando , Ludovico Paula , Sousa Maria João , Steensma H. Yde , Côrte-Real Manuela , Leão Cecília
Published: January 2003

Zygosaccharomyces bailii
ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of
Z. bailii
. This new set of
Z. bailii
strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.
Enumeration of Bifidobacteria in Gastrointestinal Samples from Piglets
Authors: Mikkelsen Lene Lind , Bendixen Christian , Jakobsen Mogens , Jensen Bent Borg
Published: January 2003

The population of
Bifidobacterium
spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new
Bifidobacterium
species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.
Effect of Associated Bacteria on the Growth and Toxicity of
Alexandrium catenella
Authors: Uribe Paulina , Espejo Romilio T.
Published: January 2003

Saprophytic bacteria in cultures of the marine dinoflagellate
Alexandrium catenella
were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of
A. catenella
disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na
+
channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of
A. catenella
are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.