bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Analysis of the
rfb
O Antigen Gene Cluster of
Salmonella enterica
Serogroup O:6,14 and Development of a Serogroup-Specific PCR Assay
Authors: Fitzgerald Collette , Sherwood Rachel , Gheesling Linda L. , Brenner Frances W. , Fields Patricia I. 
Published: October 2003

ABSTRACT

The Kauffmann-White scheme for serotyping
Salmonella
recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of
Salmonella
, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in
Salmonella.
The majority of the enzymes involved in O antigen biosynthesis are encoded by the
rfb
gene cluster. We report the sequencing of the
rfb
region from
S. enterica
serotype Sundsvall (serogroup O:6,14). The
S. enterica
serotype Sundsvall
rfb
region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized
rfb
regions, the serogroup O:6,14 sequence is most related to serogroup C
1
. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (
S. enterica
serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the
rfb
gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative
wzx
(O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of
Salmonella
serogroup O:6,14.
Escherichia coli
Ghost Production by Expression of Lysis Gene
E
and Staphylococcal Nuclease
Authors: Haidinger W. , Mayr U. B. , Szostak M. P. , Resch S. , Lubitz W. 
Published: October 2003

ABSTRACT

The production of bacterial ghosts from
Escherichia coli
is accomplished by the controlled expression of phage φX174 lysis gene
E
and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in
E. coli
resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene
E
and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.
Marker Rescue Studies of the Transfer of Recombinant DNA to
Streptococcus gordonii
In Vitro, in Foods and Gnotobiotic Rats
Authors: Kharazmi Mitra , Sczesny Silke , Blaut Michael , Hammes Walter P. , Hertel Christian 
Published: October 2003

ABSTRACT

A plasmid marker rescue system based on restoration of the
nptII
gene was established in
Streptococcus gordonii
to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10
−6
to 5.8 × 10
−7
transformants per
nptII
gene. Using a 792-bp amplification product of
nptII
the efficiency was strongly decreased (9.8 × 10
−9
). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10
−10
), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10
−11
. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of
S. gordonii
LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.
Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph,
Burkholderia cepacia
TM1, on Vanillic Acid
Authors: Mitsui Ryoji , Kusano Yoko , Yurimoto Hiroya , Sakai Yasuyoshi , Kato Nobuo , Tanaka Mitsuo 
Published: October 2003

ABSTRACT

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When
Burkholderia cepacia
TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the
hps
and
phi
genes from a methylotrophic bacterium,
Methylomonas aminofaciens
77a, were introduced into
B. cepacia
TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [
14
C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.
Characterization of Multiple-Substrate Utilization by Anthracene-Degrading
Mycobacterium frederiksbergense
LB501T
Authors: Wick Lukas Y. , Pasche Natacha , Bernasconi Stefano M. , Pelz Oliver , Harms Hauke 
Published: October 2003

ABSTRACT

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by
Mycobacterium frederiksbergense
LB501T under various substrate regimens. The distinct
13
C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that
M. frederiksbergense
LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of
M. frederiksbergense
LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by
M. frederiksbergense
LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.
Radioisotopic, Culture-Based, and Oligonucleotide Microchip Analyses of Thermophilic Microbial Communities in a Continental High-Temperature Petroleum Reservoir
Authors: Bonch-Osmolovskaya Elizaveta A. , Miroshnichenko Margarita L. , Lebedinsky Alexander V. , Chernyh Nikolai A. , Nazina Tamara N. , Ivoilov Valery S. , Belyaev Sergey S. , Boulygina Eugenia S. , Lysov Yury P. , Perov Alexander N. , Mirzabekov Andrei D. , Hippe Hans , Stackebrandt Erko , L'Haridon Stéphane , Jeanthon Christian 
Published: October 2003

ABSTRACTActivity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H2S liter−1day−1occurred at 60 and 80°C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH4liter−1day−1. Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the generaThermotoga,Thermoanaerobacter,Geobacillus,Petrotoga,Thermosipho, andThermococcus, the latter four being represented by new species. Except forThermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of theAquificales/Desulfurobacterium-Thermovibriocluster and of the generaDesulfurococcusandThermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.
A Model To Estimate the Optimal Sample Size for Microbiological Surveys
Authors: Altekruse S. F. , Elvinger F. , Wang Y. , Ye K. 
Published: October 2003

ABSTRACT

Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers. When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted. This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen. This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data. The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen. The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled. In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented. In the first scenario, bacterial colony sample size is estimated to correctly identify
Campylobacter
amplified restriction fragment length polymorphism types on broiler carcasses. The second scenario estimates bacterial colony sample size to correctly identify
Salmonella enterica
serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks. An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made.
Cloning and Characterization of the
xyl1
Gene, Encoding an NADH-Preferring Xylose Reductase from
Candida parapsilosis
, and Its Functional Expression in
Candida tropicalis
Authors: Lee Jung-Kul , Koo Bong-Seong , Kim Sang-Yong 
Published: October 2003

ABSTRACT

Xylose reductase (XR) is a key enzyme in
d
-xylose metabolism, catalyzing the reduction of
d
-xylose to xylitol. An NADH-preferring XR was purified to homogeneity from
Candida parapsilosis
KFCC-10875, and the
xyl1
gene encoding a 324-amino-acid polypeptide with a molecular mass of 36,629 Da was subsequently isolated using internal amino acid sequences and 5′ and 3′ rapid amplification of cDNA ends. The
C. parapsilosis
XR showed high catalytic efficiency (
k
cat
/

K
m

= 1.46 s
−1
mM
−1
) for
d
-xylose and showed unusual coenzyme specificity, with greater catalytic efficiency with NADH (
k
cat
/

K
m

= 1.39 × 10
4
s
−1
mM
−1
) than with NADPH (
k
cat
/

K
m

= 1.27 × 10
2
s
−1
mM
−1
), unlike all other aldose reductases characterized. Studies of initial velocity and product inhibition suggest that the reaction proceeds via a sequentially ordered Bi Bi mechanism, which is typical of XRs.
Candida tropicalis
KFCC-10960 has been reported to have the highest xylitol production yield and rate. It has been suggested, however, that NADPH-dependent XRs, including the XR of
C. tropicalis
, are limited by the coenzyme availability and thus limit the production of xylitol. The
C. parapsilosis xyl1
gene was placed under the control of an alcohol dehydrogenase promoter and integrated into the genome of
C. tropicalis
. The resulting recombinant yeast,
C. tropicalis
BN-1, showed higher yield and productivity (by 5 and 25%, respectively) than the wild strain and lower production of by-products, thus facilitating the purification process. The XRs partially purified from
C. tropicalis
BN-1 exhibited dual coenzyme specificity for both NADH and NADPH, indicating the functional expression of the
C. parapsilosis xyl1
gene in
C. tropicalis
BN-1. This is the first report of the cloning of an
xyl1
gene encoding an NADH-preferring XR and its functional expression in
C. tropicalis
, a yeast currently used for industrial production of xylitol.
Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of
Spiroplasma citri
by Its Leafhopper Vector
Circulifer haematoceps
Authors: Duret Sybille , Berho Nathalie , Danet Jean-Luc , Garnier Monique , Renaudin Joël 
Published: October 2003

ABSTRACT

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute
Spiroplasma citri
and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into
S. citri
by using an
oriC
-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector
Circulifer haematoceps
, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 × 10
6
to 2.8 × 10
6
CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 × 10
6
to 1.4 × 10
7
CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of
S. citri
by its insect vector.
Detection of
Prochlorothrix
in Brackish Waters by Specific Amplification of
pcb
Genes
Authors: Geiß Ulrike , Bergmann Ingo , Blank Miriam , Schumann Rhena , Hagemann Martin , Schoor Arne 
Published: October 2003

ABSTRACT

Prochlorothrix hollandica
is the only filamentous chlorophyll
b
(Chl
b
)-containing oxyphotobacterium that has been found in freshwater habitats to date. Chl
b
serves as a light-harvesting pigment which is bound to special binding proteins (Pcb). Even though
Prochlorothrix
was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units. Using PCR and reverse transcription, we amplified
pcb
gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea). After sequencing, high levels of homology to the
pcbB
and
pcbC
genes of
P. hollandica
were found. Furthermore, autofluorescence of
Prochlorothrix
-like filaments that indicated that Chl
b
was present was detected in enrichment cultures prepared from the estuarine phytoplankton. The detection of Chl
b
-containing filaments, as well as the
pcb
and 16S ribosomal DNA sequences, suggests that
Prochlorothrix
is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments. The potential of using
pcb
gene detection to differentiate
Prochlorothrix
from morphologically indistinguishable species belonging to the genera
Pseudanabaena
and
Planktothrix
(
Oscillatoria
) in phytoplankton analyses is discussed.
Invasion of Spores of the Arbuscular Mycorrhizal Fungus
Gigaspora decipiens
by
Burkholderia
spp
Authors: Levy Avram , Chang Barbara J. , Abbott Lynette K. , Kuo John , Harnett Gerry , Inglis Timothy J. J. 
Published: October 2003

ABSTRACT

Burkholderia
species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic
Burkholderia
spp., including
B. vietnamiensis
,
B. cepacia
, and
B. pseudomallei
, were grown on germinating spores of the arbuscular mycorrhizal fungus
Gigaspora decipiens.
Spore lysis assays revealed that all
Burkholderia
spp. tested were able to colonize the interior of
G. decipiens
spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of
B. vietnamiensis
. Twelve percent of all spores were invaded by
B. vietnamiensis
, with an average of 1.5 × 10
6
CFU recovered from individual infected spores. Of those spores inoculated with
B. pseudomallei
, 7% were invaded, with an average of 5.5 × 10
5
CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of
G. decipiens
and the attachment of bacteria.
Burkholderia
spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of
Burkholderia
spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.
Detection of the Free-Living Forms of Sulfide-Oxidizing Gill Endosymbionts in the Lucinid Habitat (
Thalassia testudinum
Environment)
Authors: Gros Olivier , Liberge Martine , Heddi Abdelaziz , Khatchadourian Chaqué , Felbeck Horst 
Published: October 2003

ABSTRACT

Target DNA from the uncultivable
Codakia orbicularis
endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of
Codakia orbicularis
is present in the environment as a free-living uncultivable form.
Identification of Peptides That Mimic the Pertussis Toxin Binding Site on Bovine Fetuin
Authors: Bogdan John A. , Yuan Wei , Long-Rowe Karen O. , Sarwar Jawad , Brucker Eric Allen , Blake M. S. 
Published: October 2003

ABSTRACTThe introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced byBordetella pertussisthat is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med.333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation ofB. pertussisin well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem.258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.