bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis
Authors: Zhang Wei , Liu Yuan , Zheng Huabao , Yang Sheng , Jiang Weihong 
Published: September 2005

ABSTRACTIn the present study, glutaryl-7-amino cephalosporanic acid acylase fromPseudomonassp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed inEscherichia colifor activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (kcat/Km) compared to wild-type CA130. TheirKmvalues were decreased by ca. 50%, and thekcatvalues increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.
Differential Expression of the Two
kdp
Operons in the Nitrogen-Fixing Cyanobacterium
Anabaena
sp. Strain L-31
Authors: Ballal Anand , Apte Shree K. 
Published: September 2005

ABSTRACT

In several types of bacteria, the Kdp ATPase (comprising of the KdpABC complex) is an inducible, high-affinity potassium transporter that scavenges K
+
from the environment. The cyanobacterium
Anabaena
sp. strain L-31 showed the presence of not one but two distinct
kdp
operons in its genome. The
kdp1
consisted of
kdpA1B1G1C1D
genes, whereas the
kdp2
contained the
kdpA2B2G2C2
genes. Among the regulatory genes, the
kdpD
open reading frame of
Anabaena
sp. strain L-31 was truncated compared to the
kdpD
of other bacteria, whereas a
kdpE
-like gene was absent in the vicinity of the two
kdp
operons. In response to K
+
limitation (<0.05 mM external K
+
), only
kdp2
(and not
kdp1
) expression could be detected as a 5.3-kb transcript on Northern blots, indicating that
kdpA2B2G2C2
genes constitute a polycystronic operon. Unlike
E. coli
, addition of osmolytes like NaCl, or a change in pH of the medium did not enhance the
kdp
expression in
Anabaena
sp. strain L-31. Interestingly, the
Anabaena
sp. strain L-31
kdp2
operon was strongly induced in response to desiccation stress. The addition of K
+
to K
+
-starved cultures resulted in repression and degradation of
kdp2
transcripts. Our results clearly show that
kdp2
is the major
kdp
operon expressed in
Anabaena
sp. strain L-31 and may play an important role in adaptation to K
+
limitation and desiccation stress.
Plasmid Donor Affects Host Range of Promiscuous IncP-1β Plasmid pB10 in an Activated-Sludge Microbial Community
Authors: De Gelder Leen , Vandecasteele Frederik P. J. , Brown Celeste J. , Forney Larry J. , Top Eva M. 
Published: September 2005

ABSTRACT

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with
lacp
-
rfp
, while
rfp
expression was repressed in the donors by chromosomal
lacI
q
. The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the α- and γ-
Proteobacteria
. The phylogenetic diversity of transconjugants obtained in separate matings with donors
Pseudomonas putida
SM1443,
Ralstonia eutropha
JMP228, and
Sinorhizobium meliloti
RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with
S. meliloti
RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.
A Persistent, Productive, and Seasonally Dynamic Vibriophage Population within Pacific Oysters (
Crassostrea gigas
)
Authors: Comeau André M. , Buenaventura Enrico , Suttle Curtis A. 
Published: September 2005

ABSTRACT

In an effort to understand the relationship between
Vibrio
and vibriophage populations, abundances of
Vibrio
spp. and viruses infecting
Vibrio parahaemolyticus
(VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 × 10
4
to 11 × 10
4
viruses cm
−3
) occurred year round in oysters, even when
V. parahaemolyticus
was undetectable (<3 cells cm
−3
). Viruses were not detected (<10 ml
−1
) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the
V. parahaemolyticus
strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among
V. alginolyticus
,
V. natriegens
, and
V. vulnificus
. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by ∼50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on
V. parahaemolyticus
indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting
V. parahaemolyticus
in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.
Epoxide Formation on the Aromatic B Ring of Flavanone by Biphenyl Dioxygenase of
Pseudomonas pseudoalcaligenes
KF707
Authors: Han Jaehong , Kim Song-Young , Jung Jihyun , Lim Yoongho , Ahn Joong-Hoon , Kim Su-Il , Hur Hor-Gil 
Published: September 2005

ABSTRACT

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding
cis
-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from
Pseudomonas pseudoalcaligenes
KF707 showed novel monooxygenase activity by converting 2(
R
)- and 2(
S
)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4
H
-chromen-4-one), whereby the epoxide bond was formed between C2′ and C3′ on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone
cis
-2′,3′-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from
P. pseudoalcaligenes
KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.
Structure and Sequence Conservation of
hao
Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History
Authors: Bergmann David J. , Hooper Alan B. , Klotz Martin G. 
Published: September 2005

ABSTRACT

Comparison of the organization and sequence of the
hao
(hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB)
Nitrosococcus oceani
and the betaproteobacterial aAOB
Nitrosospira multiformis
and
Nitrosomonas europaea
revealed a highly conserved gene cluster encoding the following proteins:
hao
, hydroxylamine oxidoreductase;
orf2
, a putative protein;
cycA
, cytochrome
c
554
; and
cycB
, cytochrome
c
m
552
. The deduced protein sequences of HAO,
c
554
, and
c
m
552
were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-
c
-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The
c
-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes
c
m
552
, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the
Proteobacteria
. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an
orf2
gene, whereas a
c
554
gene was lacking. The conservation of the
hao
gene cluster in general and the uniqueness of the
c
554
gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.
Assessment of
cry1
Gene Contents of
Bacillus thuringiensis
Strains by Use of DNA Microarrays
Authors: Letowski Jaroslaw , Bravo Alejandra , Brousseau Roland , Masson Luke 
Published: September 2005

ABSTRACT

A single
Bacillus thuringiensis
strain can harbor numerous different insecticidal crystal protein (
cry
) genes from 46 known classes or primary ranks. The
cry1
primary rank is the best known and contains the highest number of
cry
genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (
cryArray
) to test the feasibility of using microarrays to identify the
cry
gene content of
B. thuringiensis
strains. Specific 50-mer oligonucleotide probes representing the
cry1
primary and tertiary ranks were designed based on multiple
cry
gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the
cryArray
, several well-characterized
B. thuringiensis
strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked
cry1I
probes indicated the presence of a novel
cry1I
gene. Amplification and partial sequencing of the
cry1I
gene in strains IB360 and IB429 revealed the presence of a
cry1Ia
gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new
B. thuringiensis
isolates presenting interesting insecticidal activity.
Detection of Diverse New
Francisella
-Like Bacteria in Environmental Samples
Authors: Barns Susan M. , Grow Christy C. , Okinaka Richard T. , Keim Paul , Kuske Cheryl R. 
Published: September 2005

ABSTRACT

Following detection of putative
Francisella
species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia,
Francisella tularensis
, and related species. The initial survey used 16S rRNA gene primers to detect
Francisella
species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to
Francisella
were present in one water and seven soil samples. This is the first report of the detection of
Francisella
-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of
Francisella
-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from
F. tularensis
isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of
F. tularensis
subspecies support the finding of very close relatives to known
F. tularensis
strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in
F. tularensis
-specific assays. Similarly, a potential new subspecies of
Francisella philomiragia
was identified. The majority of sequences obtained, while more similar to those of
Francisella
than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of
Francisella
and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.
Bifidobacterium longumEndogalactanase Liberates Galactotriose from Type I Galactans
Authors: Hinz Sandra W. A. , Pastink Marieke I. , van den Broek Lambertus A. M. , Vincken Jean-Paul , Voragen Alphons G. J. 
Published: September 2005

ABSTRACTA putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence ofBifidobacterium longumstrain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane.galAwas cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed inEscherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. TheKmandVmaxfor AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase fromAspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.
Genome-Wide Transcriptional Variation within and between Steady States for Continuous Growth of the HyperthermophileThermotoga Maritima
Authors: Shockley Keith R. , Scott Kevin L. , Pysz Marybeth A. , Conners Shannon B. , Johnson Matthew R. , Montero Clemente I. , Wolfinger Russell D. , Kelly Robert M. 
Published: September 2005

ABSTRACTMaltose-limited, continuous growth of the hyperthermophileThermotoga maritimaat different temperatures and dilution rates (80°C/0.25 h−1, 80°C/0.17 h−1, and 85°C/0.25 h−1) showed that transcriptome-wide variation in gene expression within mechanical steady states was minimal compared to that between steady states, supporting the efficacy of chemostat-based approaches for functional genomics studies.
S434F in NrdE Generates the Thermosensitive Phenotype of
Corynebacterium ammoniagenes
CH31 and Enhances Thermolability by Increasing the Surface Hydrophobicity of the NrdE(Ts) Protein
Authors: Elhariry Hesham M. , Meens Jochen , Stehr Matthias , Auling Georg 
Published: September 2005

ABSTRACT

The thermosensitive phenotype of strain CH31, a derivative of
Corynebacterium ammoniagenes
ATCC 6872, was allocated by cloning, sequencing, and genetic complementation to a single C→T exchange in the
nrdE
(
n
ucleotide
r
e
d
uction) gene at nucleotide 1301. Protein modeling indicates the impaired surface hydrophobicity of NrdE(Ts) due to the S434F transition.
Molecular Characterization of
Clostridium tetani
Strains by Pulsed-Field Gel Electrophoresis and Colony PCR
Authors: Plourde-Owobi Lucile , Seguin Delphine , Baudin Marie-Anne , Moste Catherine , Rokbi Bachra 
Published: September 2005

ABSTRACT

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of
Clostridium tetani
. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the
tetX
gene.
Selenite and Tellurite Reduction byShewanella oneidensis
Authors: Klonowska Agnieszka , Heulin Thierry , Vermeglio André 
Published: September 2005

ABSTRACTShewanella oneidensisMR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways.
Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR
Authors: Shan X. C. , Wolffs P. , Griffiths M. W. 
Published: September 2005

ABSTRACT
In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.
Common Receptor for
Bacillus thuringiensis
Toxins Cry1Ac, Cry1Fa, and Cry1Ja in
Helicoverpa armigera
,
Helicoverpa zea
, and
Spodoptera exigua
Authors: Hernández Carmen Sara , Ferré Juan 
Published: September 2005

ABSTRACT

Binding studies using
125
I-Cry1Ac and biotinylated Cry1Fa toxins indicate the occurrence of a common receptor for Cry1Ac, Cry1Fa, and Cry1Ja in
Helicoverpa armigera
,
Helicoverpa zea
, and
Spodoptera exigua
. Our results, along with previous binding data and the observed cases of cross-resistance, suggest that this pattern seems to be widespread among lepidopteran species.
Secretion of Recombinant Pediocin PA-1 by
Bifidobacterium longum
, Using the Signal Sequence for Bifidobacterial α-Amylase
Authors: Moon Gi-Seong , Pyun Yu-Ryang , Park Myeong Soo , Ji Geun Eog , Kim Wang June 
Published: September 2005

ABSTRACT

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into
Bifidobacterium longum
MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against
Listeria monocytogenes
and the same molecular mass as native pediocin PA-1.
Method for Isolation of
Bacteroides
Bacteriophage Host Strains Suitable for Tracking Sources of Fecal Pollution in Water
Authors: Payan Andrey , Ebdon James , Taylor Huw , Gantzer Christophe , Ottoson Jakob , Papageorgiou Georgos T. , Blanch Anicet R. , Lucena Francisco , Jofre Juan , Muniesa Maite 
Published: September 2005

ABSTRACT

Bacteriophages infecting
Bacteroides
are potentially a good tool for fecal source tracking, but different
Bacteroides
host strains are needed for different geographic areas. A feasible method for isolating
Bacteroides
host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as
Bacteroides thetaiotaomicron
, was tested in several locations in Europe with excellent performance in Southern Europe.
Calcineurin-Binding Protein Cbp1 Directs the Specificity of Calcineurin-Dependent Hyphal Elongation during Mating in
Cryptococcus neoformans
Authors: Fox Deborah S. , Heitman Joseph 
Published: September 2005

ABSTRACT

Mating and virulence of the human fungal pathogen
Cryptococcus neoformans
are controlled by calcineurin, a serine-threonine-specific calcium-activated phosphatase that is the target of the immunosuppressive drugs cyclosporine A and FK506. In previous studies, a calcineurin binding protein (Cbp1, Rcn1, Dscr1/Csp1-3/MCIP1-3) that is conserved from yeasts to humans has been identified, but whether this protein functions to regulate calcineurin activity or facilitate calcineurin function as a signaling effector has been unclear. Here we show that, like calcineurin, Cbp1 is required for mating in
C. neoformans
. By contrast, Cbp1 plays no role in promoting calcineurin-dependent growth at 37°C and is not essential for haploid fruiting. Site-directed mutagenesis studies provide evidence that tandem phosphorylation and dephosphorylation of two serine residues in the conserved SP repeat motif are critical for Cbp1 function. Epistasis analysis supports models in which Cbp1 functions coordinately with calcineurin to direct hyphal elongation during mating. Taken together, these findings provide insights into the roles of Cbp1 as an accessory subunit or effector of calcineurin-specific signaling pathways, which may be features conserved among the calcipressins to govern calcineurin signaling in immune cells, cardiomyocytes, and neurons of multicellular eukaryotes.
LaeA, a Regulator of Morphogenetic Fungal Virulence Factors
Authors: Bok Jin Woo , Balajee S. Arunmozhi , Marr Kieren A. , Andes David , Nielsen Kristian Fog , Frisvad Jens C. , Keller Nancy P. 
Published: September 2005

ABSTRACT

Opportunistic animal and plant pathogens, well represented by the genus
Aspergillus
, have evolved unique mechanisms to adapt to and avoid host defenses.
Aspergillus fumigatus
, an increasingly serious pathogen owing to expanding numbers of immunocompromised patients, causes the majority of human infections; however, an inability to identify bona fide virulence factors has impeded therapeutic advances. We show that an
A. fumigatus
mutation in a developmentally expressed transcriptional regulator (
ΔlaeA
) coordinating morphological and chemical differentiation reduces virulence in a murine model; impaired virulence is associated with decreased levels of pulmonary gliotoxin and multiple changes in conidial and hyphal susceptibility to host phagocytes ex vivo. LaeA, a conserved protein in filamentous fungi, is a developmental regulator of virulence genes and, possibly, the first antimicrobial target specific to filamentous fungi that are pathogenic to plants and animals.