bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan
Authors: Sutherland Tara D. , Horne Irene , Russell Robyn J. , Oakeshott John G. 
Published: November 2002

ABSTRACT

The gram-positive bacterium
Mycobacterium
sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a
Mycobacterium
-
Escherichia coli
shuttle vector and screened for endosulfan-degrading activity in
Mycobacterium smegmatis
, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (
esd
) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the
M. smegmatis
genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.
Genetic Changes To Optimize Carbon Partitioning between Ethanol and Biosynthesis in EthanologenicEscherichia coli
Authors: Underwood S. A. , Zhou S. , Causey T. B. , Yomano L. P. , Shanmugam K. T. , Ingram L. O. 
Published: November 2002

ABSTRACTThe production of ethanol from xylose by ethanologenicEscherichia colistrain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of nativeE. colialcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.
Multiple
Streptococcus mutans
Genes Are Involved in Biofilm Formation
Authors: Yoshida Akihiro , Kuramitsu Howard K. 
Published: November 2002

ABSTRACT

Streptococcus mutans
has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in
S. mutans
biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to
S. mutans
. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the
Bacillus subtilis lytR
homologue. Another of the biofilm-defective mutants isolated was a
yulF
homologue, which encodes a hypothetical protein of
B. subtilis
whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the
comB
gene required for competence. We therefore have constructed and examined
comACDE
null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the
sgp
and
dgk
genes may play important roles in
S. mutans
biofilm formation.
Effect of Different NADH Oxidase Levels on Glucose Metabolism by
Lactococcus lactis
: Kinetics of Intracellular Metabolite Pools Determined by In Vivo Nuclear Magnetic Resonance
Authors: Neves Ana Rute , Ramos Ana , Costa Helena , van Swam Iris I. , Hugenholtz Jeroen , Kleerebezem Michiel , de Vos Willem , Santos Helena 
Published: November 2002

ABSTRACT

Three isogenic strains of
Lactococcus lactis
with different levels of H
2
O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain
L. lactis
MG1363, a NOX
−
strain harboring a deletion of the gene coding for H
2
O-forming NADH oxidase, and a NOX
+
strain with the NADH oxidase activity enhanced by about 100-fold. A comprehensive description of the metabolic events was obtained by using
13
C nuclear magnetic resonance in vivo. The most noticeable results of this study are as follows: (i) under aerobic conditions the level of fructose 1,6-bisphosphate [Fru(1,6)P
2
] was lower than the level under anaerobic conditions, and the rate of Fru(1,6)P
2
depletion was very high; (ii) the levels of 3-phosphoglycerate and phosphoenolpyruvate were considerably enhanced under aerobic conditions and significantly lower in the NOX
−
strain; and (iii) the glycolytic flux decreased in the presence of saturating levels of oxygen, but it was not altered in response to changes in the NADH oxidase activity. In particular, the observation that the glycolytic flux was not enhanced in the NOX
+
strain indicated that glycolytic flux was not primarily determined by the level of NADH in the cell. The patterns of end products were identical for the NOX
−
and parent strains; in the NOX
+
strain the carbon flux was diverted to the production of α-acetolactate-derived compounds, and at a low pH this strain produced diacetyl at concentrations up to 1.6 mM. The data were integrated with the goal of identifying the main regulatory aspects of glucose metabolism in the presence of oxygen.
Fingerprinting Closely Related
Xanthomonas
Pathovars with Random Nonamer Oligonucleotide Microarrays
Authors: Kingsley Mark T. , Straub Timothy M. , Call Douglas R. , Daly Don S. , Wunschel Sharon C. , Chandler Darrell P. 
Published: November 2002

ABSTRACT

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related
Xanthomonas
pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of
X. oryzae
strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.
Identification and Characterization of anN-Acylhomoserine Lactone-Dependent Quorum-Sensing System inPseudomonas putidaStrain IsoF
Authors: Steidle Anette , Allesen-Holm Marie , Riedel Kathrin , Berg Gabriele , Givskov Michael , Molin Søren , Eberl Leo 
Published: November 2002

ABSTRACTRecent reports have shown that several strains ofPseudomonas putidaproduceN-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promotingP. putidastrain IsoF that, when present intrans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes:ppuIandppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region betweenppuIandppuRwith significant homology torsaLfromPseudomonas aeruginosa, and a gene, designatedppuA,present upstream ofppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptionalppuA::luxABfusion we demonstrate that expression ofppuAis AHL dependent. Furthermore, transcription of the AHL synthaseppuIis shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking theppugene cluster indicated that the four genes form an island in thesuhB-PA3819 intergenic region of the currently sequencedP. putidastrain KT2440. Moreover, we provide evidence that theppugenes are not present in other AHL-producingP. putidastrains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both appuIand appuAmutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development.
Sensitivity of
Listeria monocytogenes
to Sanitizers Used in the Meat Processing Industry
Authors: Romanova Nadya , Favrin Stacy , Griffiths Mansel W. 
Published: November 2002

ABSTRACT

Nineteen
Listeria monocytogenes
strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of
L. monocytogenes
strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the
mdrL
gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.
Sakacin G, a New Type of Antilisterial Bacteriocin
Authors: Simon L. , Fremaux C. , Cenatiempo Y. , Berjeaud J. M. 
Published: November 2002

ABSTRACT

Sakacin G is a 37-amino-acid-residue-long class IIa bacteriocin produced by
Lactobacillus sake
2512, which is encoded by the duplicated structural genes
skgA1
and
skgA2
. Sakacin G appears to be unique and seems to be an intermediate between pediocin-like bacteriocins, according to its double-disulfide bridges required for antimicrobial activity, and mesentericin-like bacteriocins in terms of sequence homologies, inhibition spectrum, and specific activity.
Competitive PCR for Quantitation of a
Cytophaga-Flexibacter-Bacteroides
Phylum Bacterium Associated with the
Tuber borchii
Vittad. Mycelium
Authors: Barbieri Elena , Riccioni Giulia , Pisano Anna , Sisti Davide , Zeppa Sabrina , Agostini Deborah , Stocchi Vilberto 
Published: November 2002

ABSTRACT

An uncultured bacterium associated with the ectomycorrhizal fungus
Tuber borchii
Vittad. was identified as a novel member of the
Cytophaga-Flexibacter-Bacteroides
group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10
6
cells per 30-day-old
T. borchii
mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.
Characterization of
rpoB
Mutations in Rifampin-Resistant Clinical Isolates of
Mycobacterium tuberculosis
from Turkey by DNA Sequencing and Line Probe Assay
Authors: Cavusoglu Cengiz , Hilmioglu Suleyha , Guneri Sevinc , Bilgic Altinay 
Published: November 2002

ABSTRACT

Mutations in an 81-bp region of the
rpoB
gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of
Mycobacterium tuberculosis
isolated in Turkey. Fourteen different
rpoB
alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.
Differences in Virulence Factors among Clinical Isolates of
Escherichia coli
Causing Cystitis and Pyelonephritis in Women and Prostatitis in Men
Authors: Ruiz Joaquim , Simon Karine , Horcajada Juan P. , Velasco Maria , Barranco Margarita , Roig Gloria , Moreno-Martínez Antonio , Martínez Jose A. , Jiménez de Anta Teresa , Mensa Josep , Vila Jordi 
Published: November 2002

ABSTRACT

Differences in the presence of nine urovirulence factors among clinical isolates of
Escherichia coli
causing cystitis and pyelonephritis in women and prostatitis in men have been studied. Hemolysin and necrotizing factor type 1 occur significantly more frequently among isolates causing prostatitis than among those causing cystitis (
P
< 0.0001) or pyelonephritis (
P
< 0.005). Moreover, the
papGIII
gene occurred more frequently in
E. coli
isolates associated with prostatitis (27%) than in those associated with pyelonephritis (9%) (
P
< 0.05). Genes encoding aerobactin and PapC occurred significantly less frequently in isolates causing cystitis than in those causing prostatitis (
P
< 0.01 and
P
< 0.0001, respectively) and pyelonephritis (
P
< 0.01 and
P
< 0.0001, respectively). No differences in the presence of Sat or type 1 fimbriae were found. Finally, AAFII and Bfp fimbriae are no longer considered uropathogenic virulence factors since they were not found in any of the strains analyzed. Overall, the results showed that clinical isolates producing prostatitis need greater virulence than isolates producing pyelonephritis in women or, in particular, cystitis in women (
P
< 0.05). Overall, the results suggest that clinical isolates producing prostatitis are more virulent that those producing pyelonephritis or cystitis in women.
Evaluation of the Makromed Dipstick Assay versus PCR for Diagnosis of
Plasmodium falciparum
Malaria in Returned Travelers
Authors: Richardson David C. , Ciach Michele , Zhong Kathleen J. Y. , Crandall Ian , Kain Kevin C. 
Published: November 2002

ABSTRACT

Microscopy has been the traditional reference standard for malaria diagnosis. However, difficulty in maintaining the required technical skills, particularly in areas where malaria is not endemic, has prompted the development of rapid nonmicroscopic diagnostic assays based on the detection of malaria parasite antigen in whole blood. In this study, we evaluated the performance of one such device, the Makromed dipstick test, blindly compared to PCR and microscopy for the diagnosis of
Plasmodium falciparum
malaria in 200 febrile returned travelers. The Makromed assay detects the presence of
P. falciparum
-specific histidine-rich protein II with an antigen capture immunochromatographic strip format. Compared to PCR as the reference standard, the dipstick assay had a sensitivity of 97.0% and a specificity of 96.0%. The positive and negative predictive values were 81.2% and 99.5%, respectively. Rapid malaria diagnostic devices may provide a useful diagnostic adjunct in a clinical setting.
Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens
Authors: Manzara Stefania , Muscillo Michele , La Rosa Giuseppina , Marianelli Cinzia , Cattani Paola , Fadda Giovanni 
Published: November 2002

ABSTRACTEnterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5′ noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.
Typing of
Neisseria gonorrhoeae
Reveals Rapid Reinfection in Rural South Africa
Authors: Moodley Prashini , Martin Iona M. C. , Ison Catherine A. , Sturm A. Willem 
Published: November 2002

ABSTRACT

A recent study afforded us the opportunity to collect pre- and post-treatment isolates of
Neisseria gonorrhoeae
from women who supposedly failed to eradicate the organism when tested 8 to 10 days following treatment with a single, directly observed 250-mg dose of ciprofloxacin. In an attempt to differentiate true treatment failure from reinfection, we determined the ciprofloxacin MICs and performed auxotyping, serotyping, and opa typing of the pre- and post-treatment isolates. Paired isolates of
N. gonorrhoeae
were obtained from seven different women, despite susceptibility of the initial isolates to ciprofloxacin. Six of seven patients were infected with gonococcal isolates that differed significantly from their primary isolate. These most probably represent reinfection with a different strain, which could originate from the same partner infected with multiple strains or reinfected with a new strain or from a different partner. The susceptibility to ciprofloxacin of all isolates makes the possibility of multiple strains in the patient unlikely. The diversity of the isolates within the pairs therefore suggests rapid reinfection within the partnerships.
Campylobacter jejuniin Black-Headed Gulls (Larus ridibundus): Prevalence, Genotypes, and Influence onC. jejuniEpidemiology
Authors: Broman T. , Palmgren H. , Bergström S. , Sellin M. , Waldenström J. , Danielsson-Tham M.-L. , Olsen B. 
Published: November 2002

ABSTRACTCampylobacteriosis is a zoonotic disease in which birds have been suggested to play an important role as a reservoir. We investigated the prevalence ofCampylobacter jejunisubsp. jejuniin black-headed gulls (Larus ridibundus) in southern Sweden with the aim of examining the nature ofC. jejuniinfection in this bird species. Birds were sampled in four sampling series each year during 1999 (n= 419) and 2000 (n= 365). Longitudinally sampledC. jejuniisolates from individual gulls were subjected to macrorestriction profiling (MRP) by pulsed-field gel electrophoresis to investigate the genotypical stability during the natural course of infection. Furthermore, a subset (n= 76) of black-headed gull isolates was compared to isolates from broiler chickens (n= 38) and humans (n= 56) originating from the same geographic area. We found a pronounced seasonal variation inC. jejunicarriage, with the highest rates found in late autumn. MRP similarities were higher between isolates of human and broiler chicken origin, than between those of wild bird origin and either of the other two hosts. However, identical MRPs were found in two gull isolates and one human isolate after digestion with two restriction enzymes, strongly indicating that they may have been colonized by the same clone ofC. jejuni. The MRPs most prevalent in gull isolates did not occur among isolates from humans and broiler chickens, suggesting the existence of a subpopulation ofC. jejuniadapted to species-specific colonization or environmental survival.