bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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.