bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploration of Inorganic C and N Assimilation by Soil Microbes with Time-of-Flight Secondary Ion Mass Spectrometry
Authors: Cliff John B. , Gaspar Daniel J. , Bottomley Peter J. , Myrold David D. 
Published: October 2002

ABSTRACT

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect
13
C and
15
N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (
12
C
14
N
−
) and mass 27 (
13
C
14
N
−
and
12
C
15
N
−
) ions sputtered with a Ga
+
probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative
15
N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with
15
NO
3
−
. We observed that the
15
N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the
15
N content of cells that have assimilated
15
N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.
Detection of Anatoxin-a(s) in Environmental Samples of Cyanobacteria by Using a Biosensor with Engineered Acetylcholinesterases
Authors: Devic Eric , Li Dunhai , Dauta Alain , Henriksen Peter , Codd Geoffrey A. , Marty Jean-Louis , Fournier Didier 
Published: October 2002

ABSTRACT
Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.
Enhancement of Protective Efficacy following Intranasal Immunization with Vaccine Plus a Nontoxic LTK63 Mutant Delivered with Nanoparticles
Authors: Baudner B. C. , Balland O. , Giuliani M. M. , Von Hoegen P. , Rappuoli R. , Betbeder D. , Del Giudice G. 
Published: October 2002

ABSTRACT

Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic
Escherichia coli
heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.
Analysis of Serum Cross-Reactivity and Cross-Protection Elicited by Immunization with DNA Vaccines against
Streptococcus pneumoniae
Expressing PspA Fragments from Different Clades
Authors: Miyaji Eliane N. , Ferreira Daniela M. , Lopes Alexandre P. Y. , Brandileone M. Cristina C. , Dias Waldely O. , Leite Luciana C. C. 
Published: October 2002

ABSTRACT

Streptococcus pneumoniae
is a major cause of disease, especially in developing countries, and cost-effective alternatives to the currently licensed vaccines are needed. We constructed DNA vaccines based on pneumococcal surface protein A (PspA), an antigen shown to induce protection against pneumococcal bacteremia. PspA fragments can be divided into three families, which can be subdivided into six clades, on the basis of PspA amino acid sequence divergence (S. K. Hollingshead, R. Becker, and D. E. Briles, Infect. Immun.
68:
5889-5900, 2000). Since most clinical isolates belong to family 1 or family 2, PspA fragments from members of both of these families were analyzed. Vectors encoding the complete N-terminal regions of PspAs elicited significant humoral responses, and cross-reactivity was mainly restricted to the same family. DNA vaccines encoding fusions between PspA fragments from family 1 and family 2 were also constructed and were able to broaden the cross-reactivity, with induction of antibodies that showed reactions with members of both families. At least for the pneumococcal strains tested, the cross-reactivity of antibodies was not reflected in cross-protection. Animals immunized with DNA vaccines expressing the complete N-terminal regions of PspA fragments were protected only against intraperitoneal challenge with a strain expressing PspA from the same clade.
Lack of Strand Bias in UV-Induced Mutagenesis inEscherichia coli
Authors: Gawel Damian , Maliszewska-Tkaczyk Magdalena , Jonczyk Piotr , Schaaper Roel M. , Fijalkowska Iwona J. 
Published: October 2002

ABSTRACTWe have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in thelacZgene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of thelacZgene with respect to the origin of DNA replication. Depending on this orientation, anylacZtarget sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis inuvrAstrains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on theEscherichia colichromosome may occur with equal or similar probability on leading and lagging strands.
The Superantigen GeneypmIs Located in an Unstable Chromosomal Locus ofYersinia pseudotuberculosis
Authors: Carnoy Christophe , Floquet Stephanie , Marceau Michael , Sebbane Florent , Haentjens-Herwegh Stephanie , Devalckenaere Annie , Simonet Michel 
Published: October 2002

ABSTRACTYersinia pseudotuberculosisproduces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA,ypmB, andypmC) have been described. These genes are not found in allY. pseudotuberculosisstrains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that theypmgenes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked theypmgene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3family, was characterized downstream of theypmAandypmCgenes. A 13.3-kb region containing theypmgenes was not found in the genome ofY. pestis(CO92 and KIM 5 strains). We experimentally induced deletion of theypmgene from a superantigen-expressingY. pseudotuberculosis: using the association ofaph(3′)-IIIaandsacBgenes, we demonstrated that when these reporter genes were present in theypmlocus, deletion of these genes was about 250 times more frequent than when they were located in another region of theY. pseudotuberculosischromosome. These results indicate that unlike other superantigenic toxin genes, theYersinia ypmgenes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.
The Phage Proteomic Tree: a Genome-Based Taxonomy for Phage
Authors: Rohwer Forest , Edwards Rob 
Published: October 2002

ABSTRACTThere are ∼1031phage in the biosphere, making them the most abundant biological entities on the planet. Despite their great numbers and ubiquitous presence, very little is known about phage biodiversity, biogeography, or phylogeny. Information is limited, in part, because the current ICTV taxonomical system is based on culturing phage and measuring physical parameters of the free virion. No sequence-based taxonomic systems have previously been established for phage. We present here the “Phage Proteomic Tree,” which is based on the overall similarity of 105 completely sequenced phage genomes. The Phage Proteomic Tree places phage relative to both their near neighbors and all other phage included in the analysis. This method groups phage into taxa that predicts several aspects of phage biology and highlights genetic markers that can be used for monitoring phage biodiversity. We propose that the Phage Proteomic Tree be used as the basis of a genome-based taxonomical system for phage.
Growth Phase-Dependent Regulation of Target Gene Promoters for Binding of the Essential Orphan Response Regulator HP1043 of
Helicobacter pylori
Authors: Delany Isabel , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo 
Published: October 2002

ABSTRACT

Helicobacter pylori
encodes three two-component systems and two orphan response regulators (RRs) that are predicted to be involved in transcriptional regulation. The HP1043 gene encodes an essential OmpR-like RR, 1043RR, for which no histidine kinase has been identified. Gel filtration and cross-linking experiments on the purified 1043RR protein reveals that this protein is a dimer and in vivo dimerization assays localize the dimerization to the N-terminal regulatory domain. DNA-binding studies have revealed two targets for specific binding of the 1043RR protein and moreover, phosphorylation of the protein was not needed for the activation of binding. Footprinting analysis demonstrated that the 1043RR protein binds to its own promoter, P
1043
, overlapping the −35 promoter element from positions −17 to −45, suggesting that this protein is autoregulatory. In addition, it binds at a similar location, spanning nucleotides from positions −22 to −51 at the promoter of the methyl-accepting chemotaxis
tlpB
gene, P

tlpB

. A possible inverted repeat was identified in the binding sites of both promoters. In an attempt to overexpress 1043RR in
H. pylori
, the 10-fold induction in transcription of a second copy of HP1043 with use of an inducible promoter failed to increase cellular levels of the RR protein, suggesting that 1043RR is tightly regulated at a posttranscriptional level. The P
1043
and P

tlpB

promoters were demonstrated to be coordinately regulated in response to growth phase in
H. pylori
. The essential role of HP1043 in encoding a cell cycle regulator is discussed.
SpiC Is Required for Translocation ofSalmonellaPathogenicity Island 2 Effectors and Secretion of Translocon Proteins SseB and SseC
Authors: Freeman Jeremy A. , Rappl Catherine , Kuhle Volker , Hensel Michael , Miller Samuel I. 
Published: October 2002

ABSTRACTTheSalmonellapathogenicity island 2 (SPI2) type III secretion system (TTSS) promotesSalmonella entericaserovar Typhimurium virulence for mice and increased survival and replication within eukaryotic cells. After phagocytosis,Salmonellaserovar Typhimurium assembles the SPI2 TTSS to translocate over a dozen effector proteins across the phagosome membrane. SpiC has been previously shown to be a translocated effector with a large contribution to virulence (K. Uchiya, M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman, EMBO J. 18:3924-3933, 1999). This report demonstrates by competitive index that the virulence phenotype of aspiCmutant is equivalent to that of a secretion component mutant. In addition, translocation of SPI2 effector proteins was shown to require SpiC. Thus, the severe virulence phenotype resulting from deletion ofspiCis likely due to the inability to translocate all SPI2 effectors. SpiC was also required to secrete translocon proteins SseB and SseC but not translocated effector SseJ, indicating that lack of assembly of the translocon explains thespiCmutant phenotype.
The MexJK Efflux Pump of
Pseudomonas aeruginosa
Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan
Authors: Chuanchuen Rungtip , Narasaki Craig T. , Schweizer Herbert P. 
Published: October 2002

ABSTRACT

Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible
Pseudomonas aeruginosa
strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the
mexJK
operon. Expression of this operon is negatively regulated by the product of
mexL
, a gene located upstream of and transcribed divergently from
mexJK
. The triclosan-resistant mutant contained a single nucleotide change in
mexL
, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.
Role of ppGpp in
rpoS
Stationary-Phase Regulation in
Escherichia coli
Authors: Hirsch Matthew , Elliott Thomas 
Published: October 2002

ABSTRACT

The bacterial sigma factor RpoS is strongly induced under a variety of stress conditions and during growth into stationary phase. Here, we used
rpoS-lac
fusions in
Escherichia coli
to investigate control acting at the level of RpoS synthesis, which is especially evident when cells approach stationary phase in rich medium. Previous work has shown that the small molecule ppGpp is required for normal levels of RpoS in stationary phase. Despite the attraction of a model in which the ppGpp level controls stationary-phase induction of RpoS, careful measurement of
rpoS-lac
expression in a mutant lacking ppGpp showed similar effects during both exponential growth and stationary phase; the main effect of ppGpp was on basal expression. In addition, a modest regulatory defect was associated with the mutant lacking ppGpp, delaying the time at which full expression was achieved by 2 to 3 h. Deletion analysis showed that the defect in basal expression was distributed over several sequence elements, while the regulatory defect mapped to the region upstream of the
rpoS
ribosome-binding site (RBS) that contains a
cis
-acting antisense element. A number of other genes that have been suggested as regulators of
rpoS
were tested, including
dksA
,
dsrA
,
barA
,
ppkx
, and
hfq
. With the exception of the
dksA
mutant, which had a modest defect in Luria-Bertani medium, none of these mutants was defective for
rpoS
stationary-phase induction. Even a short
rpoS
segment starting at 24 nucleotides upstream of the AUG initiation codon was sufficient to confer substantial stationary-phase regulation, which was mainly posttranscriptional. The effect of RBS-proximal sequence was independent of all known
trans
-acting factors, including ppGpp.
Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex
Authors: Cho Eun Hee , Gumport Richard I. , Gardner Jeffrey F. 
Published: October 2002

ABSTRACT
Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.
Vaginal
Lactobacillus
Flora of Healthy Swedish Women
Authors: Vásquez Alejandra , Jakobsson Tell , Ahrné Siv , Forsum Urban , Molin Göran 
Published: October 2002

ABSTRACT

Species of the
Lactobacillus acidophilus
complex are generally considered to constitute most of the vaginal
Lactobacillus
flora, but the flora varies between studies. However, this may be due to difficulties in identifying the closely related species within the
L. acidophilus
complex by using traditional methods and to variations in the vaginal status of the participants. Two hundred two isolates from the vaginal fluids of 23 Swedish women without bacterial vaginosis, as defined by the criteria of Nugent et al. (R. P. Nugent, M. A. Krohn, and S. L. Hillier, J. Clin. Microbiol. 29:297-301, 1991), were typed by randomly amplified polymorphic DNA (RAPD) analysis and identified to the species level by temporal temperature gradient gel electrophoresis, multiplex PCR, and 16S ribosomal DNA sequencing. The vaginal flora of most participants was dominated by a single RAPD type, but five of them harbored two RAPD types representing two different species or strains. The most frequently occurring species were
Lactobacillus crispatus
,
Lactobacillus gasseri
,
Lactobacillus iners
, and
Lactobacillus jensenii. L. iners
has not previously been reported as one of the predominant
Lactobacillus
species in the vagina.
Fluorescent Amplified Fragment Length Polymorphism Probabilistic Database for Identification of Bacterial Isolates from Urinary Tract Infections
Authors: Kassama Yankuba , Rooney Paul J. , Goodacre Royston 
Published: October 2002

ABSTRACT

The ability of the fluorescent amplified fragment length polymorphism (FAFLP) technique to identify bacterial isolates from urinary tract infections (UTIs) was investigated. FAFLP was carried out using the single primer combination
Mse
I plus CT and
Eco
RI plus 0, and information-rich FAFLP profiles were generated from all 69 UTI isolates studied, which comprised both gram-negative and gram-positive bacteria encompassing eight genera. The genetic relatedness of these 69 bacteria was determined by cluster analysis, and this revealed eight main groups corresponding to the eight bacterial genera. Finer discrimination on the same dendrogram showed species and subspecies differentiations, thus demonstrating the potential of FAFLP for describing a wide diversity range within microbial populations. The interpretation of FAFLP profiles is often complicated because it relies upon the investigator interpreting dendrograms; this process may be subjective if the tree is complicated, particularly if it includes polytomies (unresolved nodes). Therefore, we have developed a method based on Bayes' theorem for the identification of bacteria against an FAFLP probabilistic identification matrix. Thus, FAFLP is suitable for the objective identification of causal agents of UTI, and the procedure offers great potential in the clinical laboratory.
Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR
Authors: Luo Guizhen , Mitchell Thomas G. 
Published: October 2002

ABSTRACT

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify
Candida albicans
,
Candida glabrata
,
Candida parapsilosis
,
Candida tropicalis
, and
Aspergillus fumigatus
. Another set of previously published ITS primers, CN4 and CN5, were used to identify
Cryptococcus neoformans
. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of
Aspergillus
, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.