bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain ofKlebsiella pneumoniae
Authors: Yigit Hesna , Queenan Anne Marie , Anderson Gregory J. , Domenech-Sanchez Antonio , Biddle James W. , Steward Christine D. , Alberti Sebastian , Bush Karen , Tenover Fred C. 
Published: July 2002

ABSTRACTAKlebsiella pneumoniaeisolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence ofblaSHVandblaTEMgenes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia colishowed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniaecarbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,blaKPC-1, was cloned inE. coliand shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescensS6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parentK. pneumoniaestrain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance inK. pneumoniaestrain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.
Resistance of
Streptococcus pneumoniae
to Deformylase Inhibitors Is Due to Mutations in
defB
Authors: Margolis Peter , Hackbarth Corinne , Lopez Sara , Maniar Mita , Wang Wen , Yuan Zhengyu , White Richard , Trias Joaquim 
Published: July 2002

ABSTRACT

Resistance to peptide deformylase inhibitors in
Escherichia coli
or
Staphylococcus aureus
is due to inactivation of transformylase activity. Knockout experiments in
Streptococcus pneumoniae
R6x indicate that the transformylase (
fmt
) and deformylase (
defB
) genes are essential and that a
def
paralog (
defA
) is not. Actinonin-resistant mutants of
S. pneumoniae
ATCC 49619 harbor mutations in
defB
but not in
fmt
. Reintroduction of the mutated
defB
gene into wild-type
S. pneumoniae
R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
Sulfonamide Resistance in
Haemophilus influenzae
Mediated by Acquisition of
sul2
or a Short Insertion in Chromosomal
folP
Authors: Enne Virve I. , King Anna , Livermore David M. , Hall Lucinda M. C. 
Published: July 2002

ABSTRACT

Determinants of sulfonamide resistance were investigated in clinical isolates of
Haemophilus influenzae
from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in
H. influenzae
has not previously been reported. Eight isolates requiring at least 1,024 μg of sulfamethoxazole per ml for inhibition carried the
sul2
gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase,
folP
, was found to carry an insertion of 15 bp together with other missense mutations relative to
folP
of
H. influenzae
strain Rd RM118 (MIC, 8 μg/ml); the
folP
sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 μg/ml (although these exhibited considerable divergence in
folP
sequence) and in highly resistant isolates carrying
sul2
. Transformation with a 599-bp fragment of
folP
containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by
sul2
and modified
folP
, were detected in isolates from both the United Kingdom and Kenya.
Use of Combined Microautoradiography and Fluorescence In Situ Hybridization To Determine Carbon Metabolism in Mixed Natural Communities of Uncultured Bacteria from the Genus
Achromatium
Authors: Gray N. D. , Howarth R. , Pickup R. W. , Jones J. Gwyn , Head I. M. 
Published: July 2002

ABSTRACT

Combined microautoradiography and fluorescence in situ hybridization (FISH) was used to investigate carbon metabolism in uncultured bacteria from the genus
Achromatium
. All of the
Achromatium
species identified in a freshwater sediment from Rydal Water, Cumbria, United Kingdom, which were distinguishable only by FISH, assimilated both [
14
C]bicarbonate and [
14
C]acetate. This extends previous findings that
Achromatium
spp. present at another location could only utilize organic carbon sources.
Achromatium
spp., therefore, probably exhibit a range of physiologies, i.e., facultative chemolithoautotrophy, mixotrophy, and chemoorganoheterotrophy, similar to other large sulfur bacteria (e.g.,
Beggiatoa
spp.).
Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of
Clonostachys rosea
and Development of a Strain-Specific PCR Detection Assay
Authors: Bulat Sergey A. , Lübeck Mette , Alekhina Irina A. , Jensen Dan Funck , Knudsen Inge M. B. , Lübeck Peter Stephensen 
Published: July 2002

ABSTRACT

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of
Clonostachys rosea
(syn.
Gliocladium roseum
) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.
xylP
Promoter-Based Expression System and Its Use for Antisense Downregulation of the
Penicillium chrysogenum
Nitrogen Regulator NRE
Authors: Zadra Ivo , Abt Beate , Parson Walther , Haas Hubertus 
Published: July 2002

ABSTRACT

A highly inducible fungal promoter derived from the
Penicillium chrysogenum
endoxylanase (
xylP
) gene is described. Northern analysis and the use of a β-glucuronidase (
uidA
) reporter gene strategy showed that
xylP
expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the
niaD
locus in
P. chrysogenum
and at the
argB
locus in
Aspergillus nidulans
demonstrated that the
xylP
promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the
Aspergillus
species. The
xylP
promoter was found to be 65-fold more efficient than the isopenicillin-
N
-synthetase (
pcbC
) promoter in
Penicillium
and 23-fold more efficient than the nitrate reductase (
niaD
) promoter in
Aspergillus
under induced conditions. Furthermore, the
xylP
promoter was used for controllable antisense RNA synthesis of the
nre
-encoded putative major nitrogen regulator of
P. chrysogenum
. This approach led to inducible downregulation of the steady-state mRNA level of
nre
and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of
nreB
, which encodes a negative-acting nitrogen regulatory GATA factor of
Penicillium
, was found to be subject to regulation by NRE. Our data are the first direct evidence that
nre
indeed encodes an activator in the nitrogen regulatory circuit in
Penicillium
and indicate that cross regulation of the controlling factors occurs.
Genetic Linkage Map of the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Pérez Gúmer , Ritter Enrique , Pisabarro Antonio G. , Ramı́rez Lucı́a 
Published: July 2002

ABSTRACT

We have constructed a genetic linkage map of the edible basidiomycete
Pleurotus ostreatus
(var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (
est1
); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of
P. ostreatus
, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of
P. ostreatus
.
Coordinated, Differential Expression of Two Genes through Directed mRNA Cleavage and Stabilization by Secondary Structures
Authors: Smolke Christina D. , Carrier Trent A. , Keasling J. D. 
Published: July 2002

ABSTRACT

Metabolic engineering and multisubunit protein production necessitate the expression of multiple genes at coordinated levels. In bacteria, genes for multisubunit proteins or metabolic pathways are often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to place multiple genes under the control of a single promoter and produce proteins encoded in that novel operon in different ratios over a range of inducer concentrations. RNase E sites identified in the
Rhodobacter capsulatus puf
operon and
Escherichia coli pap
operon were separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5′ and 3′ ends of the coding regions. The introduced RNase E site directed cleavage between the coding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease cleavage by engineered 3′ secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5′ end. The relative expression levels of two reporter genes could be varied up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the ribosome binding site, this technology should allow one to create new operons and coordinate, yet separately control, the expression levels of genes expressed in that operon.
The
Bradyrhizobium japonicum
Proline Biosynthesis Gene
proC
Is Essential for Symbiosis
Authors: King Natalie D. , Hojnacki David , O'Brian Mark R. 
Published: July 2002

ABSTRACT

Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The
Bradyrhizobium japonicum proC
gene was isolated, and a
proC
mutant strain that behaved as a strict proline auxotroph in culture was constructed. The
proC
strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the
proC
gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.
A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk
Authors: Gunasekera Thusitha S. , Attfield Paul V. , Veal Duncan A. 
Published: July 2002

ABSTRACT

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (≤1-h) FCM assay based on enzymatic clearing of milk to determine total bacteria in milk. When bacteria were added to ultra-heat-treated milk, a good correlation (
r
≥ 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (
P
< 0.01) and a good agreement (
r
= 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was ≤10
4
bacteria ml of milk
−1
. This limit is below the level of detection required to satisfy legislation in many countries and states.
Efficient Heterologous Expression in
Aspergillus oryzae
of a Unique Dye-Decolorizing Peroxidase, DyP, of
Geotrichum candidum
Dec 1
Authors: Sugano Yasushi , Nakano Ryosuke , Sasaki Katsuya , Shoda Makoto 
Published: July 2002

ABSTRACT

Efficient expression of the dye-decolorizing peroxidase, DyP, from
Geotrichum candidum
Dec 1 in
Aspergillus oryzae
M-2-3 was achieved by fusing mature cDNA encoding
dyp
with the
A. oryzae
α-amylase promoter (
amyB
). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in
A. oryzae
. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by
A. oryzae
exhibits a function similar to that of native DyP.
Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity
Authors: Mason Maria G. , Ball Andrew S. , Reeder Brandon J. , Silkstone Gary , Nicholls Peter , Wilson Michael T. 
Published: July 2002

ABSTRACT

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete
Thermomonospora fusca
BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete
Streptomyces viridosporus
T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.
Identification of Bacteria in Pasteurized Zucchini Purées Stored at Different Temperatures and Comparison with Those Found in Other Pasteurized Vegetable Purées
Authors: Guinebretiere Marie-Hélène , Berge Odile , Normand Philippe , Morris Cindy , Carlin Frédéric , Nguyen-The Christophe 
Published: July 2002

ABSTRACT

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25°C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial
rrs
gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the
Bacillus circulans
(group 2) API taxon or in taxa with a low number of positive API characters such as
Brevibacillus brevis
. A phylogenetic analysis pointed to one new species of
Bacillus
and three new species of
Paenibacillus
among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as
B. circulans
(group 2) or
B. circulans/B. macerans/B.
polymyxa
were assigned to a new
Paenibacillus
taxon phylogenetically related to
P. azotofixans
. Storage conditions at 4°C favored the development of “
B. macroides/B. maroccanus
” and
Paenibacillus
spp. in zucchini purées and
Paenibacillus
spp. in other purées. Storage conditions at 20 to 25°C favored the development of
B. subtilis
group (
B. licheniformis
and
B. subtilis
) and
B. cereus
group strains. At 10°C,
Paenibacillus
spp. were always present at high frequencies, whereas the occurrence of
B. macroides/B. maroccanus
(in zucchini purées),
B. cereus
, and
B. pumilus
varied with the experiment.
Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria
Authors: Allen Andrew E. , Booth Melissa G. , Frischer Marc E. , Verity Peter G. , Zehr Jonathan P. , Zani Sabino 
Published: July 2002

ABSTRACT

A PCR approach was used to construct a database of
nasA
genes (called
narB
genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A
nasA
-specific PCR primer set that could be used to selectively amplify the
nasA
gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced
nasA
genes. Our results indicate that several groups of heterotrophic bacterial
nasA
genes are common and widely distributed in oceanic environments.
Effects of the 20-Kilodalton Helper Protein on Cry1Ac Production and Spore Formation in
Bacillus thuringiensis
Authors: Shao Zongze , Liu Ziduo , Yu Ziniu 
Published: July 2002

ABSTRACT

Bacillus thuringiensis
produces large amounts of various pesticidal proteins during the stationary phase. In order to achieve a high yield and form crystals, some pesticidal proteins require the presence of other proteins. Helper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in
B. thuringiensis
subsp.
israelensis
. Although full-length Cry1 protoxins are usually independent in terms of expression and crystallization in
B. thuringiensis
, in this study P20 significantly enhanced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and plasmid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 μm long and 0.85 μm wide, three times the size of the crystals formed in the control lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against
Heliothis armigera
larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was observed, which has seldom been reported previously. Actually, most protein was completely degraded during synthesis, and after synthesis about one-third of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indicating that it acted as a molecular chaperon. In addition, spores were smaller and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synthesis; this degradation was effectively relieved by P20, which resulted in enhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.
Efficient Homolactic Fermentation by
Kluyveromyces lactis
Strains Defective in Pyruvate Utilization and Transformed with the Heterologous
LDH
Gene
Authors: Bianchi Michele M. , Brambilla Luca , Protani Francesca , Liu Chi-Li , Lievense Jefferson , Porro Danilo 
Published: July 2002

ABSTRACT

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a
Kluyveromyces lactis
yeast strain lacking the single pyruvate decarboxylase gene (
KlPDC1
) and transformed with the heterologous lactate dehydrogenase gene (
LDH
). The
LDH
gene was placed under the control the
KlPDC1
promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g
−1
, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used
K. lactis LDH
transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g
−1
(maximum theoretical yield, 1 g g
−1
), and with high LDH activity.