bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines
Authors: Collis Christina M. , Kim Mi-Jurng , Partridge Sally R. , Stokes H. W. , Hall Ruth M. 
Published: July 2002

ABSTRACTIntegrons capture gene cassettes by using a site-specific recombination mechanism. As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined. The configuration of the three potentially definitive features of integrons, theintI3gene, the adjacentattI3recombination site, and the Pcpromoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5′ conserved segment) of class 1 integrons. The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encodingtniRgene on the other, suggesting that they are part of a transposable element related to Tn402but with the integron module in the opposite orientation. The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, theattI3site in the integron, and different cassette-associated 59-be (59-base element) sites. Both integration of circularized cassettes into theattI3site and excision of integrated cassettes were also catalyzed by IntI3. TheattI3site was localized to a short region adjacent to theintI3gene. Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.
The InhA2 Metalloprotease of
Bacillus thuringiensis
Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route
Authors: Fedhila Sinda , Nel Patricia , Lereclus Didier 
Published: July 2002

ABSTRACT

The entomopathogenic bacterium
Bacillus thuringiensis
is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the
inhA
gene, named
inhA2
, in
B. thuringiensis
strain 407 Cry
−
. The
inhA2
gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional
inhA2
′-
lacZ
fusion to show that
inhA2
expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of
inhA2
suggests that Spo0A directly regulates the transcription of
inhA2
. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized
Bombyx mori
larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against
Galleria mellonella
larvae infected via the oral pathway. The spores of Δ
inhA2
mutant strain were ineffective in providing synergism whereas those of the Δ
inhA
mutant strain were not. These results indicate that the
B. thuringiensis
InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.
Phage TP901-1 Site-Specific Integrase Functions in Human Cells
Authors: Stoll Stephanie M. , Ginsburg Daniel S. , Calos Michele P. 
Published: July 2002

ABSTRACT

We demonstrate that the site-specific integrase encoded by phage TP901-1 of
Lactococcus lactis
subsp.
cremoris
has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in
Escherichia coli
and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in
E. coli
and determined that for complete reaction in
E. coli,
the minimal sizes of
attB
and
attP
are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.
Molecular Cloning and Expression of Mn
2+
-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium,
Pseudomonas
sp. Strain TK4
Authors: Sueyoshi Noriyuki , Kita Katsuhiro , Okino Nozomu , Sakaguchi Keishi , Nakamura Takashi , Ito Makoto 
Published: July 2002

ABSTRACT

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium,
Pseudomonas
sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn
2+
. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from
Bacillus cereus
,
Staphylococcus aureus
,
Listeria ivanovii
, and
Leptospira interrogans
, as well as a hypothetical protein encoded in
Chromobacterium violaceum
, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
VmrA, a Member of a Novel Class of Na
+
-Coupled Multidrug Efflux Pumps from
Vibrio parahaemolyticus
Authors: Chen Jing , Morita Yuji , Huda M. Nazmul , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa 
Published: July 2002

ABSTRACT

Gene
vmrA
, cloned from
Vibrio parahaemolyticus
, made
Escherichia coli
resistant to 4prime;,6-diamino-2-phenylindol, tetraphenylphosphonium chloride, acriflavine, and ethidium bromide. VmrA belongs to the DinF branch of MATE family efflux transporters. VmrA catalyzed acriflavine efflux and showed Na
+
/drug transporter activity because the addition of tetraphenylphosphonium to Na
+
-loaded cells caused Na
+
efflux.
FimZ Is a Molecular Link between Sticking and Swimming in
Salmonella enterica
Serovar Typhimurium
Authors: Clegg Steven , Hughes Kelly T. 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Typhimurium produces two types of filamentous appendages on its surface. Fimbriae mediate adherence to tissues and cells via receptor-specific interactions, and flagella are the organelles of motility. These appendages play a role in colonization and dissemination, respectively, from infected surfaces and may be important components of bacterial survival. Increased expression of FimZ in serovar Typhimurium resulted in bacteria which were hyperfimbriated but were nonmotile in soft agar. This lack of motility was associated with down regulation of the
flhDC
master flagellar operon. Therefore, FimZ represents a molecular connection between flagella and fimbrial formation in serovar Typhimurium, indicating that the synthesis of flagella and fimbriae are oppositely controlled.
Endotoxin of
Neisseria meningitidis
Composed Only of Intact Lipid A: Inactivation of the Meningococcal 3-Deoxy-
d
-Manno-Octulosonic Acid Transferase
Authors: Tzeng Yih-Ling , Datta Anup , Kolli V. Kumar , Carlson Russell W. , Stephens David S. 
Published: July 2002

ABSTRACT

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-
d
-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar
kdtA
::
aphA-3
mutation was created in
Neisseria meningitidis
via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the
kdtA
::
aphA-3
mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV
A
did not accumulate. The LOS structure of the
kdtA
::
aphA-3
mutant was restored to the wild-type structure by complementation with
kdtA
from
N. meningitidis
or
Escherichia coli
. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in
N. meningitidis
can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.
Characterization of a
Mycobacterium intracellulare
Variant Strain by Molecular Techniques
Authors: Menendez M. C. , Palenque E. , Navarro M. C. , Nuñez M. C. , Rebollo M. J. , Garcia M. J. 
Published: July 2002

ABSTRACT

This paper describes a
Mycobacterium intracellulare
variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the
Mycobacterium avium
complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as
mig
(macrophage-induced gene). The results obtained identify the isolates as a variant of
M. intracellulare
, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members.
New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections
Authors: Harder Timm C. , Hufnagel Markus , Zahn Katrin , Beutel Karin , Schmitt Heinz-Josef , Ullmann Uwe , Rautenberg Peter 
Published: July 2002

ABSTRACT
Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically ≥5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.
Mycobacterium interjectum
as Causative Agent of Cervical Lymphadenitis
Authors: De Baere Thierry , Moerman Mieke , Rigouts Leen , Dhooge Catharina , Vermeersch Hubert , Verschraegen Gerda , Vaneechoutte Mario 
Published: July 2002

ABSTRACT

A mycobacterial strain isolated from a lymph node of a 3-year-old female with cervical lymphadenitis was identified as
Mycobacterium interjectum
by means of sequencing of the 16S rRNA gene. Analysis of this case and previously published cases demonstrates the importance of
M. interjectum
as a causative agent of cervical lymphadenitis in young children.
Isolation of Amantadine-Resistant Influenza A Viruses (H3N2) from Patients following Administration of Amantadine in Japan
Authors: Iwahashi Jun , Tsuji Katsuro , Ishibashi Tetsuya , Kajiwara Junboku , Imamura Yoshihiro , Mori Ryoichi , Hara Koyu , Kashiwagi Takahito , Ohtsu Yasushi , Hamada Nobuyuki , Maeda Hisao , Toyoda Michiko , Toyoda Tetsuya 
Published: July 2002

ABSTRACT
In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.
Does Blood of Healthy Subjects Contain Bacterial Ribosomal DNA?
Authors: Nikkari Simo , McLaughlin Ian J. , Bi Wanli , Dodge Deborah E. , Relman David A. 
Published: July 2002

ABSTRACT
Real-time PCR methods with primers and a probe targeting conserved regions of the bacterial 16S ribosomal DNA (rDNA) revealed a larger amount of rDNA in blood specimens from healthy individuals than in matched reagent controls. However, the origins and identities of these blood-associated bacterial rDNA sequences remain obscure.
Evaluation of a Reformulated CHROMagar Candida
Authors: Jabra-Rizk Mary Ann , Brenner Troy M. , Romagnoli Mark , Baqui A. A. M. A. , Merz William G. , Falkler William A. , Meiller Timothy F. 
Published: July 2002

ABSTRACT

CHROMagar Candida is a differential culture medium for the isolation and presumptive identification of clinically important yeasts. Recently the medium was reformulated by Becton Dickinson. This study was designed to evaluate the performance of the new formula of CHROMagar against the original CHROMagar Candida for recovery, growth, and colony color with stock cultures and with direct plating of clinical specimens. A total of 90 stock yeast isolates representing nine yeast species, including
Candida dubliniensis
, as well as 522 clinical specimens were included in this study. No major differences were noted in growth rate or colony size between the two media for most of the species. However, all 10
Candida albicans
isolates evaluated consistently gave a lighter shade of green on the new CHROMagar formulation. In contrast, all 26
C. dubliniensis
isolates gave the same typical dark green color on both media. A total of 173 of the 522 clinical specimens were positive for yeast, with eight yeast species recovered. The recovery rates for each species were equivalent on both media, with no consistent species-associated differences in colony size or color. Although both media were comparable in performance, the lighter green colonies of
C. albicans
isolates on the new CHROMagar made it easier to differentiate between
C. albicans
and
C. dubliniensis
isolates. In conclusion, the newly formulated Becton Dickinson CHROMagar Candida medium is as equally suited as a differential medium for the presumptive identification of yeast species and for the detection of multiple yeast species in clinical specimens as the original CHROMagar Candida medium.
Serosubtypes and PorA Types of
Neisseria meningitidis
Serogroup B Isolated in Brazil during 1997-1998: Overview and Implications for Vaccine Development
Authors: Sacchi Claudio T. , Lemos Ana Paula S. , Popovic Tanja , Cassio de Morais Jose , Whitney Anne M. , Melles Carmo Elias A. , Brondi Luciana M. G. , Monteiro Lucia M. C. , Paiva Maria V. , Solari Claude A. , Mayer Leonard W. 
Published: July 2002

ABSTRACT

Meningococcal disease caused by
N. meningitidis
serogroup B (MenB) has been endemic in Brazil since 1997. In this study, we determined the prevalence of serosubtypes of MenB isolated in 10 Brazilian states and the Federal District during 1997 and 1998 and investigated the extent of PorA VR sequence variation among the most prevalent serosubtypes to evaluate the possible use of an outer membrane vesicle (OMV)-, PorA-based vaccine to prevent meningococcal disease in Brazil. During this period, a total of 8,932 cases of meningococcal disease were reported. Only 42% (
n
= 3,751) of the reported cases were laboratory confirmed, and about 60% (
n
= 2,255) of those were identified as MenB. Among 1,297 MenB strains selected for this study, the most prevalent serosubtypes were P1.19,15 (66%), P1.7,1 (11%), and P1.7,16 (4%). PorA VR typing showed that 91% of the P1.19,15 strains analyzed had VR1 and VR2 sequences identical to those of the prototype strain. No sequence variation was detected among the 40 strains representing all isolated MenB P1.7,16 strains in the three southern states, where this serosubtype accounts for 75% of the serosubtypes identified. Similarly, all P1.7,1 strains were identified by PorA typing as P1.7-1,1. Although further improvements in the reporting of cases and collection of strains in Brazil are needed, our data suggest that a trivalent OMV-based vaccine prepared with PorA types P1.19,15, P1.7-1,1, and P1.7,16 may be appropriate to control serogroup B meningococcal disease in most of the Brazilian states.
Identification and Characterization of Variable-Number Tandem Repeats in the
Yersinia pestis
Genome
Authors: Klevytska Alexandra M. , Price Lance B. , Schupp James M. , Worsham Patricia L. , Wong Jane , Keim Paul 
Published: July 2002

ABSTRACT

Yersinia pestis
, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the
Y. pestis
genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the
Y. pestis
genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (
R
= 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.
Diagnosis of Placental Malaria
Authors: Mockenhaupt Frank P. , Ulmen Ulrike , von Gaertner Christiane , Bedu-Addo George , Bienzle Ulrich 
Published: July 2002

ABSTRACT

In a group of 596 delivering Ghanaian women, the sensitivities of peripheral blood thick film microscopy, ICT Malaria P.f/P.v test, and PCR in detecting microscopically confirmed placental
Plasmodium falciparum
infection were 42, 80, and 97%, respectively. In addition to the gross underestimation of placental malaria by peripheral blood film microscopy, submicroscopic infections were found to be a risk factor for maternal anemia.
Geographic Distribution of Mating Type Alleles of
Cryptococcus neoformans
in Four Areas of the United States
Authors: Yan Zhun , Li Xiaogang , Xu Jianping 
Published: July 2002

ABSTRACT

To better understand the epidemiology and population structure of
Cryptococcus neoformans
, we determined mating types for 358
C. neoformans
strains isolated through the active surveillance program from 1992 to 1994 in four geographic areas in the United States: San Francisco, California; Georgia; Texas; and Alabama. Two assays were used to determine mating types: (i) crossing with standard laboratory tester strains JEC20 and JEC21 on V8 agar medium; and (ii) PCR with the mating type α allele-specific primer of the
STE12
gene and with serotype (A and D)- and mating type (a and α)-specific primers of the
STE20
gene. Using these two methods, we found that this sample consisted of the following: (i) 324 serotype A, mating type (
MAT
) α (Aα) strains; (ii) 12 serotype D, α (Dα) strains; (iii) 14 serotype AD strains with mating type alleles Aa and Dα (AaDα); (iv) 2 serotype AD strains with mating type alleles Aα and Da (AαDa); (v) 3 serotype B, α (Bα) strains; and (vi) 3 serotype AD strains but with only one mating type allele. No strain with
MAT
a was found within serotype A, B, or D in this collection. Interestingly, 14 of the 19 serotype AD strains contained the Aa allele at the
STE20
locus; 13 of these 14 were from San Francisco. Our results suggest that the environment in San Francisco might contain Aa strains capable of mating with Dα strains. In addition, our result demonstrate that the sample from San Francisco had a significantly higher proportion of self-fertile strains than those from the other three areas.
Prospective Evaluation of the New Chromogenic Medium Candida ID, in Comparison with Candiselect, for Isolation of Molds and Isolation and Presumptive Identification of Yeast Species
Authors: Letscher-Bru Valerie , Meyer Marie-Helene , Galoisy Anne-Cecile , Waller Jocelyn , Candolfi Ermanno 
Published: July 2002

ABSTRACT

We conducted a prospective evaluation of Candida ID chromogenic medium (bioMérieux, Marcy l'Etoile, France) with 786 clinical specimens in comparison with Candiselect medium (Bio-Rad, Marnes la Coquette, France). Candida ID chromogenic medium identified 97.7% of
Candida albicans
strains; enabled presumptive identification of
C. tropicalis
,
C. lusitaniae
,
C. guillermondii
, and
C. kefyr
and better detection of yeast combinations (11.4% more often); and was more sensitive for the isolation of filamentous fungi (17.7% more often). However, Candida ID chromogenic medium appeared to be less selective vis-à-vis bacteria, with bacterial colonies sometimes pigmented blue.
Comparison of Chromogenic and Fluorogenic Membrane Filtration Methods for Detection of Four
Candida
Species
Authors: Bauters T. G. , Nelis H. J. 
Published: July 2002

ABSTRACT

CHROMagar Candida medium was inoculated with a variety of clinical samples (
n
= 282) by using membrane filtration instead of swabbing or streaking. This modified CHROMagar procedure increased the ability to detect yeast by 16.7%. Compared to an enzymatic two-step membrane filtration method with fluorogenic substrates, it yielded similar recovery but was slower.