bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Authors: Meroth Christiane B. , Walter Jens , Hertel Christian , Brandt Markus J. , Hammes Walter P. 
Published: January 2003

ABSTRACT

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few
Lactobacillus
species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour),
Lactobacillus sanfranciscensis
and a new species,
L
.
mindensis
, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively
L
.
crispatus
and
L
.
pontis
became the predominant species in sourdough B and
L
.
crispatus
,
L
.
panis
, and
L
.
frumenti
became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran),
L
.
johnsonii
and
L
.
reuteri
were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which
L
.
fermentum
was also detected. Isolates of the species
L
.
sanfranciscensis
and
L
.
fermentum
were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTGenetic 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

ABSTRACTAlfalfa 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

ABSTRACTIn 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.