bluuebunny/crossref_metadata_2025 huggingface dataset:
SIR1, an Upstream Component in Auxin Signaling Identified by Chemical Genetics
Authors: Zhao Yunde , Dai Xinhua , Blackwell Helen E. , Schreiber Stuart L. , Chory Joanne
Published: August 2003

Auxin is a plant hormone that regulates many aspects of plant growth and development. We used a chemical genetics approach to identify
SIR1
, a regulator of many auxin-inducible genes. The
sir1
mutant was resistant to sirtinol, a small molecule that activates many auxin-inducible genes and promotes auxin-related developmental phenotypes.
SIR1
is predicted to encode a protein composed of a ubiquitin-activating enzyme E1–like domain and a Rhodanese-like domain homologous to that of prolyl isomerase. We suggest a molecular context for how the auxin signal is propagated to exert its biological effects.
Discovery of Gene Function by Expression Profiling of the Malaria Parasite Life Cycle
Authors: Le Roch Karine G. , Zhou Yingyao , Blair Peter L. , Grainger Muni , Moch J. Kathleen , Haynes J. David , De la Vega Patricia , Holder Anthony A. , Batalov Serge , Carucci Daniel J. , Winzeler Elizabeth A.
Published: August 2003

The completion of the genome sequence for
Plasmodium falciparum
, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only ∼35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying
Plasmodium
parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.
A MicroRNA as a Translational Repressor of
APETALA2
in
Arabidopsis
Flower Development
Authors: Chen Xuemei
Published: August 2003

Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their target messenger RNAs. Here, Ishow that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene,
APETALA2
, regulates
APETALA2
expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects similar to those in loss-of-function
apetala2
mutants. Elevated levels of mutant
APETALA2
RNA with disrupted miRNA172 base pairing, but not wild-type
APETALA2
RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of
APETALA2
in
Arabidopsis
flower development.
Characterization of the RpoS Status of Clinical Isolates of
Salmonella enterica
Authors: Robbe-Saule Véronique , Algorta Gabriela , Rouilhac Isabelle , Norel Françoise
Published: August 2003

The stationary-phase-inducible sigma factor, σ
S
(RpoS), is the master regulator of the general stress response in
Salmonella
and is required for virulence in mice.
rpoS
mutants can frequently be isolated from highly passaged laboratory strains of
Salmonella
. We examined the
rpoS
status of 116 human clinical isolates of
Salmonella
, including 41
Salmonella enterica
serotype Typhi strains isolated from blood, 38
S. enterica
serotype Typhimurium strains isolated from blood, and 37
Salmonella
serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ
S
protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type
rpoS
gene. Our results showed that 15 of the 41
Salmonella
serotype Typhi isolates were defective in RpoS. We sequenced the
rpoS
allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ
S
, showing that the
rpoS
mutations are not clonal. Thus, mutant
rpoS
alleles can be found in freshly isolated clinical strains of
Salmonella
serotype Typhi, and they may affect virulence properties. Interestingly however, no
rpoS
mutants were found among the 75
Salmonella
serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the
rpoS
status. This suggests that
Salmonella
serotype Typhimurium
rpoS
mutants are counterselected because
rpoS
plays a role in the pathogenesis of
Salmonella
serotype Typhimurium in humans or in the transmission cycle of the disease.
Cloning of a Nitrilase Gene from the CyanobacteriumSynechocystissp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein
Authors: Heinemann Ute , Engels Dirk , Bürger Sibylle , Kiziak Christoph , Mattes Ralf , Stolz Andreas
Published: August 2003

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacteriumSynechocystissp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein inEscherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.
Microbial and Physiological Characterization of Weakly Amylolytic but Fast-Growing Lactic Acid Bacteria: a Functional Role in Supporting Microbial Diversity in Pozol, a Mexican Fermented Maize Beverage
Authors: Díaz-Ruiz G. , Guyot J. P. , Ruiz-Teran F. , Morlon-Guyot J. , Wacher C.
Published: August 2003

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping;
Streptococcus bovis
strains were found to be predominant.
Streptococcus macedonicus
,
Lactococcus lactis
, and
Enterococcus sulfureus
strains were also identified.
S. bovis
strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight]
−1
) and specific rate of amylase production (130.7 U g [cell dry weight]
−1
h
−1
). In contrast, it showed a high specific growth rate (0.94 h
−1
) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate
−1
). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.
Comparing the Dehalogenase Gene Pool in Cultivated α-Halocarboxylic Acid-Degrading Bacteria with the Environmental Metagene Pool
Authors: Marchesi Julian R. , Weightman Andrew J.
Published: August 2003

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for α-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II
deh
gene was greater than that of the group I
deh
gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.
Overexpression of the Gene Encoding GTP:Mannose-1-Phosphate Guanyltransferase,
mpg1
, Increases Cellular GDP-Mannose Levels and Protein Mannosylation in
Trichoderma reesei
Authors: Zakrzewska Anna , Palamarczyk Grazyna , Krotkiewski Hubert , Zdebska Ewa , Saloheimo Markku , Penttilä Merja , Kruszewska Joanna S.
Published: August 2003

To elucidate the regulation and limiting factors in the glycosylation of secreted proteins, the
mpg1
and
dpm1
genes from
Trichoderma reesei
(
Hypocrea jecorina
) encoding GTP:α
-d-
mannose-1-phosphate guanyltransferase and dolichyl phosphate mannose synthase (DPMS), respectively, were overexpressed in
T. reesei
. No significant increases were observed in DPMS activity or protein secretion in
dpm1
-overexpressing transformants, whereas overexpression of
mpg1
led to a twofold increase in GDP-mannose (GDPMan) levels. GDPMan was effectively utilized by mannnosyltransferases and resulted in hypermannosylation of secreted proteins in both N and O glycosylation. Overexpression of the
mpg1
gene also increased the transcription of the
dpm1
gene and DPMS activity. Our data indicate that the level of cellular GDPMan can play a major regulatory role in protein glycosylation in
T. reesei
.
Vibrio harveyi
Nitroreductase Is Also a Chromate Reductase
Authors: Kwak Young Hak , Lee Dong Seok , Kim Han Bok
Published: August 2003

The chromate reductase purified from
Pseudomonas ambigua
was found to be homologous with several nitroreductases.
Escherichia coli
DH5α and
Vibrio harveyi
KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione
S
-transferase (GST) and
E. coli
DH5α NfsA (GST-EcNfsA), a fusion between GST and
E. coli
DH5α NfsB (GST-EcNfsB), and a fusion between GST and
V. harveyi
KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The

K
m

values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The
V
max
values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each
V
max
/

K
m

value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to
E. coli
than Cr(VI).
Rhizobium leguminosarum
Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants
Authors: Ma Wenbo , Guinel Frèdèrique C. , Glick Bernard R.
Published: August 2003

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by
Rhizobium
to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia,
Rhizobium leguminosarum
bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (
acdS
) and its regulatory gene, a leucine-responsive regulatory protein-like gene (
lrpL
), were constructed and tested to determine their abilities to nodulate
Pisum sativum
L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in
R. leguminosarum
bv. viciae 128C53K enhances the nodulation of
P. sativum
L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by
Rhizobium
to promote nodulation by adjusting ethylene levels in legumes.
Elucidation of the Transmission Patterns of an Insect-Borne Bacterium
Authors: Darby A. C. , Douglas A. E.
Published: August 2003

Quantitative data on modes of transmission are a crucial element in understanding the ecology of microorganisms associated with animals. We investigated the transmission patterns of a γ-proteobacterium informally known as pea aphidBemisia-like symbiont (PABS), also known as T-type, which is widely but not universally distributed in natural populations of the pea aphid,Acyrthosiphon pisum. The vertical transmission of PABS to asexual and sexual morphs and sexually produced eggs was demonstrated by a diagnostic PCR-based assay, and the maximum estimated failure rate was 2%. Aphids naturally lacking PABS acquired PABS bacteria administered via the diet, and the infection persisted by vertical transmission for at least three aphid generations. PABS was also detected in two of five aphid honeydew samples tested and in all five siphuncular fluid samples tested but in none of 15 samples of salivary secretions from PABS-positive aphids. However, PABS-negative aphids did not acquire PABS when they were cocultured with PABS-positive aphids; the maximal estimated level of horizontal transmission was 18%. A deterministic model indicated that the force of infection by a horizontal transmission rate of 3% is sufficient to maintain a previously described estimate of the prevalence of PABS-positive aphids (37%), if the vertical transmission rate is 98%. We concluded that PABS infections inA. pisumcan be maintained by high vertical transmission rates and occasional horizontal transmission, possibly via the oral route, in the absence of selection either for or against aphids bearing this bacterium.
Application of the
shsp
Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria
Authors: El Demerdash Hassan A. M. , Heller Knut J. , Geis Arnold
Published: August 2003

Plasmid pSt04 of
Streptococcus thermophilus
contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the
shsp
plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned
shsp
gene into
S. thermophilus
St11 lacking a plasmid encoding
shsp
resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for
S. thermophilus
, based on the plasmid-encoded
shsp
gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant
shsp
plasmids into different
S. thermophilus
and
Lactococcus lactis
strains. Using a recombinant plasmid carrying an erythromycin resistance (Em
r
) gene in addition to
shsp
, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in
S. thermophilus
(when we were selecting for heat resistance) were determined to be 2.4 × 10
4
and 1.0 × 10
4
CFU/0.5 μg of DNA, with standard deviations of 0.54 × 10
4
and 0.32 × 10
4
, for
shsp
and Em
r
selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 10
4
and 3.8 × 10
3
CFU/0.5 μg of DNA, with standard deviations of 0.63 × 10
4
and 3.48 × 10
3
, for
shsp
and Em
r
selection, respectively. The applicability of
shsp
as a selection marker was further demonstrated by constructing
S. thermophilus
plasmid pHRM1 carrying the
shsp
gene as a selection marker and the restriction-modification genes of another
S. thermophilus
plasmid as a functional trait.
Multiplex Real-Time PCR for Monitoring
Heterobasidion annosum
Colonization in Norway Spruce Clones That Differ in Disease Resistance
Authors: Hietala Ari M. , Eikenes Morten , Kvaalen Harald , Solheim Halvor , Fossdal Carl G.
Published: August 2003

A multiplex real-time PCR assay was developed to monitor the dynamics of the
Picea abies
-
Heterobasidion annosum
pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.
A New Bacterial Steroid Degradation Gene Cluster in
Comamonas testosteroni
TA441 Which Consists of Aromatic-Compound Degradation Genes for Seco-Steroids and 3-Ketosteroid Dehydrogenase Genes
Authors: Horinouchi Masae , Hayashi Toshiaki , Yamamoto Takako , Kudo Toshiaki
Published: August 2003

In
Comamonas testosteroni
TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the
meta-
cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17,
tesI
,
tesH
, ORF11, ORF12, and
tesDEFG.
TesH and TesI are 3-ketosteroid-Δ
1
-dehydrogenase and 3-ketosteroid-Δ
4
(5α)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9α-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.
Differences in Susceptibility of
Listeria monocytogenes
Strains to Sakacin P, Sakacin A, Pediocin PA-1, and Nisin
Authors: Katla T. , Naterstad K. , Vancanneyt M. , Swings J. , Axelsson L.
Published: August 2003

Two hundred strains of
Listeria monocytogenes
collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin. The individual 50% inhibitory concentrations (IC
50
) were determined in a microtiter assay and expressed in nanograms per milliliter. The IC
50
of sakacin P ranged from 0.01 to 0.61 ng ml
−1
. The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml
−1
, respectively. The use of a large number of strains and the accuracy of the IC
50
determination revealed patterns not previously described, and for the first time it was shown that the IC
50
of sakacin P divided the
L. monocytogenes
strains into two distinct groups. Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism. The results from these studies essentially confirmed the grouping based on the IC
50
of sakacin P. A high correlation was found between the IC
50
of sakacin P and that of pediocin PA-1 for the 200 strains. Surprisingly, the correlation between the IC
50
of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC
50
of sakacin A and the class I bacteriocin nisin.
Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of
Candida magnoliae
Authors: Lee Jung-Kul , Koo Bong-Seong , Kim Sang-Yong , Hyun Hyung-Hwan
Published: August 2003

Mannitol biosynthesis in
Candida magnoliae
HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from
C. magnoliae
HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of
C. magnoliae
MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively.
C. magnoliae
MDH showed high substrate specificity and high catalytic efficiency (
k
cat
= 823 s
−1
,
K

m

= 28.0 mM, and
k

cat

/
K

m

= 29.4 mM
−1
s
−1
) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and
C. magnoliae
MDH is specific for transferring the 4-
pro
-
S
hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of
C. magnoliae
MDH showed a significant homology with SDRs from various sources, indicating that the
C. magnoliae
MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources,
C. magnoliae
MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes
C. magnoliae
MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.
Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of
Hansenula polymorpha
Authors: Kim So-Young , Sohn Jung-Hoon , Bae Jung-Hoon , Pyun Yu-Ryang , Agaphonov Michael O. , Ter-Avanesyan Michael D. , Choi Eui-Sung
Published: August 2003

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in
Hansenula polymorpha
. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of
H. polymorpha
, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of
H. polymorpha
provides a suitable library for activity-based selection of mutants after directed evolution.
Spread of Recombinant DNA by Roots and Pollen of Transgenic Potato Plants, Identified by Highly Specific Biomonitoring Using Natural Transformation of an
Acinetobacter
sp
Authors: de Vries Johann , Heine Martin , Harms Klaus , Wackernagel Wilfried
Published: August 2003

Transgenic potato plants with the
nptII
gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of
nptII
with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of
Acinetobacter
sp. strain BD413 cells having in their genomes a terminally truncated
nptII
gene (
nptII
′; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in
nptII
′ and tg4 restored
nptII
, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring
nptII
in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4°C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.
Diversity and Seasonal Variability of β-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning
Authors: Brümmer I. H. M. , Felske A. , Wagner-Döbler I.
Published: August 2003

The β-subgroup of the
Proteobacteria
has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for β-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described βI cluster of cosmopolitan freshwater bacteria, where they represented new species related to
Rhodoferax
,
Aquaspirillum
, and
Hydrogenophaga.
RBFs 4 to 7 affiliated with
Aquabacterium commune
,
Ideonella dechloratans
, and
Sphaerotilus natans
, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to
Gallionella ferruginea
. Seasonal changes in the relative intensity of the β-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.
Role of Capsular Colanic Acid in Adhesion of Uropathogenic
Escherichia coli
Authors: Hanna Andrea , Berg Michael , Stout Valerie , Razatos Anneta
Published: August 2003

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic
Escherichia coli
strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic
E. coli
and inert substrates.
Production and Properties of Bacteriocin-Like Inhibitory Substances from the Swine Pathogen
Streptococcus suis
Serotype 2
Authors: Mélançon D. , Grenier D.
Published: August 2003

Streptococcus suis
serotype 2 is a major pathogen found in the upper respiratory tract of swine. In this study, isolates of this bacterial species were tested for the production of bacteriocin-like inhibitory substances (BLIS). Of the 38 strains tested, four inhibited the growth of other
S. suis
isolates according to a deferred-antagonism plate assay. Interestingly, three of the strains were originally isolated from healthy carrier pigs and were considered nonvirulent. Three isolates (94-623, 90-1330, and AAH4) that produced BLIS in liquid broth were selected for further characterization. None of the inhibitory activities was related to the production of either organic acids or hydrogen peroxide. The BLIS produced by these strains were heat stable and proteinase K, pronase, and elastase sensitive but were trypsin and chymotrypsin resistant. They were stable at pH 2 and 12 and had molecular masses in the range of 14 to 30 kDa. Maximum production was observed during the mid-log phase. Following a curing procedure with novobiocin, only 90-1330 lost the ability to produce BLIS, suggesting that the BLIS might be plasmid encoded. Analysis of the inhibitory spectra revealed that the BLIS-producing strains also inhibited the growth of
Actinobacillus minor
,
Actinobacillus porcinus
,
Enterococcus durans
,
Micrococcus luteus
,
Streptococcus agalactiae
,
Streptococcus dysgalactiae subsp. dysgalactiae
,
Streptococcus equi
subsp.
zooepidemicus
, and
S. dysgalactiae subsp. equisimilis
. This study reports for the first time the ability of the swine pathogen
S. suis
serotype 2 to produce BLIS with the characteristics of classic bacteriocins. Further studies are required to investigate the possibility of using bacteriocin-producing strains to prevent swine infections caused by virulent strains of
S. suis
serotype 2.
Preslaughter Holding Environment in Pork Plants Is Highly Contaminated withSalmonella enterica
Authors: Rostagno M. H. , Hurd H. S. , McKean J. D. , Ziemer C. J. , Gailey J. K. , Leite R. C.
Published: August 2003

The objective of this study was to determine whether abattoir pens can provide aSalmonella entericainfection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied (∼150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period (∼2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification ofS. entericaby primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated withS. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive forS. enterica. All 24 groups of pigs hadS. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 differentSalmonellaserovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point forSalmonellacontamination in the preharvest pork production chain.
Molecular Characterization of Microsporidia Indicates that Wild Mammals Harbor Host-Adapted
Enterocytozoon
spp. as well as Human-Pathogenic
Enterocytozoon bieneusi
Authors: Sulaiman Irshad M. , Fayer Ronald , Lal Altaf A. , Trout James M. , Schaefer Frank W. , Xiao Lihua
Published: August 2003

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of
Enterocytozoon
spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of
Enterocytozoon
spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described
Enterocytozoon bieneusi
genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic
E
.
bieneusi
. However, there are also host-adapted
Enterocytozoon
genotypes in wildlife, which may represent species different from
E
.
bieneusi
and have no apparent public health significance. This is the first report of
E
.
bieneusi
in wildlife.
PCR Assay of the
groEL
Gene for Detection and Differentiation of
Bacillus cereus
Group Cells
Authors: Chang Yu-Hsiu , Shangkuan Yung-Hui , Lin Hung-Chi , Liu Hwan-Wun
Published: August 2003

Strains of species in the
Bacillus cereus
group are potentially enterotoxic. Thus, the detection of all
B. cereus
group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the
B. cereus
group, we explored the potential of the
groEL
gene as a phylogenetic marker. A phylogenetic analysis of the
groEL
sequences of 78
B. cereus
group strains revealed that the
B. cereus
group strains were split into two major clusters, one including six
B. mycoides
and one
B. pseudomycoides
(cluster II) and the other including two
B. mycoides
and the rest of the
B. cereus
group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The
sodA
gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the
sodA
sequences showed substantial similarity to the tree constructed from the
groEL
sequences. Based on the
groEL
sequences, a PCR assay for detection and identification of
B. cereus
group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the
B. cereus
group strains. RFLP with
Mbo
I was identical for all the
B. cereus
group strains analyzed, while RFLP with
Mfe
I or
Pst
I classified all
B. cereus
and
B. thuringiensis
strains into two groups. All cluster II
B. mycoides
and
B. pseudomycoides
strains could be discriminated from other
B. cereus
group bacteria by restriction analysis with
Tsp
RI.
Isolation and Characterization of
Campylobacter
Bacteriophages from Retail Poultry
Authors: Atterbury Robert J. , Connerton Phillippa L. , Dodd Christine E. R. , Rees Catherine E. D. , Connerton Ian F.
Published: August 2003

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of
Campylobacter
in poultry production. To this end, we have developed a procedure to recover
Campylobacter
bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized
Campylobacter
phage (i.e., NCTC 12674). By using this method, we have shown that
Campylobacter
phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous
Campylobacter
phages. Thirty-four
Campylobacter
bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (
P
< 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that
Campylobacter
bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.
Biocontrol of
Listeria monocytogenes
on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin
Authors: Leverentz Britta , Conway William S. , Camp Mary J. , Janisiewicz Wojciech J. , Abuladze Tamuna , Yang Ming , Saftner Robert , Sulakvelidze Alexander
Published: August 2003

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied
Listeria monocytogenes
populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic,
L. monocytogenes
-specific phages via two phage application methods, spraying and pipetting, on
L. monocytogenes
populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced
L. monocytogenes
populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced
L. monocytogenes
populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced
L. monocytogenes
populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of
L. monocytogenes
.
Osmoregulation in the Parasitic Protozoan
Tritrichomonas foetus
Authors: Maroulis Sarah L. , Schofield Philip J. , Edwards Michael R.
Published: August 2003

Tritrichomonas foetus
was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of
T. foetus
intracellular amino acids revealed a pool size (65 mM) that was similar to that of
Trichomonas vaginalis
but was considerably smaller than those of
Giardia intestinalis
and
Crithidia luciliae
. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the
T. foetus
RVI. The
T. foetus
intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K
+
concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the
T. foetus
RVI was enhanced by elevated external K
+
concentrations and was inhibited when K
+
and/or Cl
−
was absent from the medium. The results suggested that the well-documented Na
+
-K
+
-2Cl
−
cotransport system was responsible for the K
+
influx activated during the RVI. However, inhibitors of Na
+
-K
+
-2Cl
−
cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K
+
influx in
T. foetus
.
Identification and Characterization of Phenylpyruvate Decarboxylase Genes in
Saccharomyces cerevisiae
Authors: Vuralhan Zeynep , Morais Marcos A. , Tai Siew-Leng , Piper Matthew D. W. , Pronk Jack T.
Published: August 2003

Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding α-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in
Saccharomyces cerevisiae
, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway.
S. cerevisiae
possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity:
YDR380w/ARO10
,
YDL080C/THI3
,
PDC1
,
PDC5
, and
PDC6
. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (
ARO10
) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that
ARO10
was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene
THI3
did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (
PDC1
,
PDC5
, and
PDC6
) for the alternative activity. The

K
m

and
V
max
values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering of the Ehrlich pathway.