bluuebunny/crossref_metadata_2025_split huggingface dataset:
Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases
Authors: van Hylckama Vlieg Johan E. T. , Tang Lixia , Lutje Spelberg Jeffrey H. , Smilda Tim , Poelarends Gerrit J. , Bosma Tjibbe , van Merode Annet E. J. , Fraaije Marco W. , Janssen Dick B. 
Published: July 2002

ABSTRACT

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in
Escherichia coli
, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of
Agrobacterium radiobacter
strain AD1, designated HheC, was purified to homogeneity. The
k
cat
and
K

m

values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s
−1
and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK
a
of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.
Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of
Escherichia coli degP
Mutants and Induces the Cpx and ς
E
Extracytoplasmic Stress Responses
Authors: Langen Geoffrey R. , Harper Jill R. , Silhavy Thomas J. , Howard S. Peter 
Published: July 2002

ABSTRACT

DegP is a periplasmic protease that is a member of both the ς
E
and Cpx extracytoplasmic stress regulons of
Escherichia coli
and is essential for viability at temperatures above 42°C. [U-
14
C]acetate labeling experiments demonstrated that phospholipids were degraded in
degP
mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from
degP
cells at the nonpermissive temperature. A mutation in
pldA
, which encodes outer membrane phospholipase A (OMPLA), was found to rescue
degP
cells from the temperature-sensitive phenotype.
pldA degP
mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants.
degP
and
pldA degP
mutants containing chromosomal
lacZ
fusions to Cpx and ς
E
regulon promoters indicated that both regulons were activated in the
pldA
mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of
degP
mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the
degP
temperature-sensitive phenotype by inducing the Cpx and ς
E
regulons rather than by inactivating the phospholipase per se.
Gene Cluster of
Rhodothermus marinus
High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a
caa
3
-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases
Authors: Santana Margarida , Pereira Manuela M. , Elias Nuno P. , Soares Cláudio M. , Teixeira Miguel 
Published: July 2002

ABSTRACT

The respiratory chain of the thermohalophilic bacterium
Rhodothermus marinus
contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named
rcoxA
to
rcoxD
) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of
rcoxD
. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the
R. marinus
enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E
278
,
Paracoccus denitrificans
numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu
A
domain, that is characteristic of
caa
3
oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this
caa
3
oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the
caa
3
class of heme-copper cytochrome
c
oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.
The C Terminus of ς
32
Is Not Essential for Degradation by FtsH
Authors: Tomoyasu Toshifumi , Arsène Florence , Ogura Teru , Bukau Bernd 
Published: July 2002

ABSTRACT

A key step in the regulation of heat shock genes in
Escherichia coli
is the stress-dependent degradation of the heat shock promoter-specific ς
32
subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including ς
32
, as degradation signals for AAA proteases. We investigated the role of the C terminus of ς
32
in FtsH-dependent degradation by analysis of C-terminally truncated ς
32
mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of ς
32
did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of ς
32
was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated ς
32
mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of ς
32
in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.
Influence of the Nucleoid on Placement of FtsZ and MinE Rings inEscherichia coli
Authors: Sun Qin , Margolin William 
Published: July 2002

ABSTRACTWe previously presented evidence that replicating but unsegregated nucleoids, along with the Min system, act as topological inhibitors to restrict assembly of the FtsZ ring (Z ring) to discrete sites in the cell. To test if nonreplicating nucleoids have similar exclusion effects, we examined Z rings indnaA(temperature sensitive) mutants. Z rings were excluded from centrally localized nucleoids and were often observed at nucleoid edges. Cells with nonreplicating nucleoids formed filaments, some of which contained large nucleoid-free areas in which Z rings were positioned at regular intervals. Because MinE may protect FtsZ from the action of the MinC inhibitor in these nucleoid-free zones, we examined the localization of a MinE-green fluorescent protein (GFP) fusion with respect to Z rings and nucleoids. Like Z rings, MinE-GFP appeared to localize independently of nucleoid position, forming rings at regular intervals in nucleoid-free regions. Unlike FtsZ, however, MinE-GFP often localized on top of nucleoids, replicating or not, suggesting that MinE is relatively insensitive to the nucleoid inhibition effect. These data suggest that both replicating and nonreplicating nucleoids are capable of topologically excluding Z rings but not MinE.
Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from
Streptococcus uberis
Authors: Ward Philip N. , Leigh James A. 
Published: July 2002

ABSTRACT

A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from
Streptococcus uberis
strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of
S. uberis
strains. Analysis of the locus normally occupied by
pauA
revealed an absence of the
pauA
open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the
pauB
gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.
Nucleotide and Amino Acid Sequences of
oriT-traM-traJ-traY-traA-traL
Regions and Mobilization of Virulence Plasmids of
Salmonella enterica
Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium
Authors: Chu Chishih , Chiu Cheng-Hsun , Chu Chi-Hong , Ou Jonathan T. 
Published: July 2002

ABSTRACT

The virulence plasmid of
Salmonella enterica
serovar Gallinarum-Pullorum (pSPV) but not those of
Salmonella enterica
serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the
oriT-traM-traJ-traY-traA-traL
regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the
oriT
of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the
oriT
of the other two pSVs. The nucleotide sequences of the
oriT-traM-traJ-traY-traA-traL
region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in
traJ
,
traY
, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus βαα fragments in the TraY of pSPV and F plasmid, whereas there was only a single βαα fragment in that of pSEV and pSTV.
Automation of Fluorescence-Based PCR for Confirmation of Meningococcal Disease
Authors: Diggle M. A. , Edwards G. F. S. , Clarke S. C. 
Published: July 2002

ABSTRACT

A fluoresence-based PCR method was developed, fully automated, and used to confirm infection with
Neisseria meningitidis
by detection of the meningococcus-specific
ctrA
gene. The method provided a highly sensitive, high-throughput assay that was reproducible and less labor-intensive than manual methods.
Mutations in the
rpoB
Gene of Rifampin-Resistant
Mycobacterium tuberculosis
Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay
Authors: Garcia Lucia , Alonso-Sanz Mercedes , Rebollo Maria J. , Tercero Juan C. , Chaves Fernando 
Published: July 2002

ABSTRACT

Genetic alterations in the
rpoB
gene were characterized in 50 rifampin-resistant (Rif
r
) clinical isolates of
Mycobacterium tuberculosis
complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of
rpoB
mutations was evaluated with isolates of the
M. tuberculosis
complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different
rpoB
mutations among the Rif
r
isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rif
r
allele within each strain, 10 of the 11 Rif
r
genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of
M. tuberculosis
complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rif
r
organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods.
Simple Method for Determining Biovar and Serovar Types of
Ureaplasma urealyticum
Clinical Isolates Using PCR–Single-Strand Conformation Polymorphism Analysis
Authors: Pitcher David , Sillis Margaret , Robertson Janet A. 
Published: July 2002

ABSTRACT

Ureaplasma urealyticum
has been associated with urethritis in men, obstetric problems in women, and respiratory distress syndrome in preterm infants.
U. urealyticum
can be divided into two biovars comprising 14 serovars. Partial sequences of genes encoding the multiple-banded antigens of the cell surface are known. Using a commercially available precast DNA mutation detection gel system, we have developed a simple and reproducible PCR–single-strand conformation polymorphism analysis method for differentiating the biovars of this species that reveals five patterns among the 14 serovars and enables clinical isolates to be typed directly from broth cultures.
Laboratory Diagnosis of
Clostridium difficile
-Associated Diarrhea and Colitis: Usefulness of Premier Cytoclone A+B Enzyme Immunoassay for Combined Detection of Stool Toxins and Toxigenic
C. difficile
Strains
Authors: Lozniewski Alain , Rabaud Christian , Dotto Emmanuelle , Weber Michele , Mory Francine 
Published: July 2002

ABSTRACT

Detection of
Clostridium difficile
toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for
C. difficile
followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of
C. difficile
-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.
Distinguishing
Candida
Species by β-
N
-Acetylhexosaminidase Activity
Authors: Niimi Kyoko , Shepherd Maxwell G. , Cannon Richard D. 
Published: July 2002

ABSTRACT

A variety of fungi produce the hydrolytic enzyme β-
N
-acetylhexosaminidase (HexNAcase), which can be readily detected in assays by using
p
-nitrophenyl-
N
-acetyl-β-
d
-glucosaminide as a substrate. In the present study we developed a microtiter plate-based HexNAcase assay for distinguishing
Candida albicans
and
Candida dubliniensis
strains from other yeast species. HexNAcase activity was detected in 89 of 92 (97%)
C. albicans
strains and 4 of 4
C. dubliniensis
strains but not in 28 strains of eight other
Candida
species, 4
Saccharomyces cerevisiae
strains, or 2
Cryptococcus neoformans
strains. The HexNAcase activity in
C. albicans
and
C. dubliniensis
was strain specific. All except three clinical
C. albicans
isolates among the
C. albicans
strains tested produced enzyme activity within 24 h. These strains did produce enzyme activity, however, after a prolonged incubation period. For two of these atypical strains, genomic DNA at the
C. albicans HEX1
gene locus, which encodes HexNAcase, showed nucleotide differences from the sequence of control strains. Among the other
Candida
species tested, only
C. dubliniensis
had a DNA sequence that hybridized with the
HEX1
probe under low-stringency conditions. The microtiter plate-based assay used in the present study for the detection of HexNAcase activity is a simple, relatively inexpensive method useful for the presumptive identification of
C. albicans
and
C. dubliniensis
.
Molecular Profiles of Group B Streptococcal Surface Protein Antigen Genes: Relationship to Molecular Serotypes
Authors: Kong Fanrong , Gowan Sonia , Martin Diana , James Gregory , Gilbert Gwendolyn L. 
Published: July 2002

ABSTRACT

The study of surface protein antigens of group B streptococci (GBS) is important for understanding of the pathogenesis and epidemiology of infection, and several of these antigens have been proposed as components of GBS conjugate vaccines. In a previous study, we developed a novel PCR-and-sequencing system for identification of GBS serotypes and serosubtypes based on the capsular polysaccharide synthesis (
cps
) gene cluster. In this study, we used published sequences to develop PCR assays for identification of genes encoding GBS surface proteins including C alpha (
bca
), C alpha-like proteins 2 and 3 (
alp2
and
alp3
), Rib (
rib
), and C beta (
bac
). We showed that the prototype R reference strain, Prague 25/60, contained a novel alpha-like protein antigen gene (the proposed
alp4
), which presumably encodes an atypical, but antigenically similar, R-like protein. Initial evaluation of these gene-specific assays showed excellent specificity. By combining
cps
serotypes, serosubtypes, and surface protein gene profiles, we were able to divide 224 GBS isolates into 31 serovariants. GBS
bac-
positive strains could be further subtyped into 11 groups and 20 subgroups. Our results confirmed and extended reported associations between some
cps
serotypes and serosubtypes, on the one hand, and surface protein genes, on the other: serosubtypes III-1 and III-2 were associated with
rib
, serosubtype III-3 with
alp2
, serotype Ib with
bca
and
bac
, and serotype V with
alp3
. The associations between serotype Ia and
bca, bca
repetitive unit, and
bca
repetitive unit-like sequence-containing genes need to be studied further. These PCR-based methods will provide an alternative and objective tool for subtyping of GBS based on surface protein antigen genes.
Comparison of Molecular and Conventional Methods for Typing of Enteroviral Isolates
Authors: Manayani Darly Joseph , Shaji R. V. , Fletcher G. John , Cherian Thomas , Murali N. , Sathish N. , Solomon Tilak , Gnanamuthu Chandran , Sridharan Gopalan 
Published: July 2002

ABSTRACT
Twenty-eight enteroviral isolates obtained from various clinical specimens were typed by Lim-Benyesh-Melnick (LBM) pool-based neutralization, PCR-restriction fragment length polymorphism (RFLP), and partial sequencing of the VP1 region of the enteroviral genome. Sequencing was found to be a good alternative to LBM typing, while PCR-RFLP was inadequate for identification of enteroviral isolates.
Evolution of the IS
6110-
Based Restriction Fragment Length Polymorphism Pattern during the Transmission of
Mycobacterium tuberculosis
Authors: Warren R. M. , van der Spuy G. D. , Richardson M. , Beyers N. , Booysen C. , Behr M. A. , van Helden P. D. 
Published: July 2002

ABSTRACT

Interpretation of the molecular epidemiological data of
Mycobacterium tuberculosis
is dependent on the validity of the assumptions that have been made. It is assumed that the IS
6110
banding pattern is sufficiently stable to define epidemiological events representing ongoing transmission. However, molecular epidemiological data also support the observation that the IS
6110
banding pattern may change over time. Factors affecting this rate may include the nature and duration of disease in a host and the opportunity to experience different host environments during the transmission cycle. To estimate the rate of IS
6110
change occurring during the process of transmission,
M. tuberculosis
isolates from epidemiologically linked patients were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis. The identification of IS
6110
banding pattern changes during ongoing transmission suggested that a rate could be estimated. IS
6110
change was significantly associated with strains with >5 IS
6110
elements (
P
= 0.013) and was not observed in low-copy-number isolates. The minimum rate of appearance of variant strains was calculated to be 0.14 variant cases per source-case per year. This data suggest that clustering of isolates based on identical RFLP patterns is expected to underestimate transmission in patients infected with high-copy-number isolates. A model based on the rate of appearance of both variant and invariant strains demonstrates that the genotypically defined population structure may change by 18.6% during the study period of approximately 6.5 years. The implications for the use of RFLP data for epidemiologic study are discussed.
Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen
Candida parapsilosis
Authors: Nosek Jozef , Tomáška L'ubomír , Ryčovská Adriana , Fukuhara Hiroshi 
Published: July 2002

ABSTRACT

Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen
Candida parapsilosis
. The strategy for identification of
C. parapsilosis
strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy.