bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

Activity 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

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

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

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

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

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

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

The 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.
Comparison of Velocity Profiles for Different Flow Chamber Designs Used in Studies of Microbial Adhesion to Surfaces
Authors: Bakker D. P. , van der Plaats A. , Verkerke G. J. , Busscher H. J. , van der Mei H. C.
Published: October 2003

Flow chambers are commonly used to study microbial adhesion to surfaces under environmentally relevant hydrodynamic conditions. The parallel plate flow chamber (PPFC) is the most common design, and mass transport occurs through slow convective diffusion. In this study, we analyzed four different PPFCs to determine whether the expected hydrodynamic conditions, which control both mass transport and detachment forces, are actually achieved. Furthermore, the different PPFCs were critically evaluated based on the size of the area where the velocity profile was established and constant with a range of flow rates, indicating that valid observations could be made. Velocity profiles in the different chambers were calculated by using a numerical simulation model based on the finite element method and were found to coincide with the profiles measured by particle image velocimetry. Environmentally relevant shear rates between 0 and 10,000 s
−1
could be measured over a sizeable proportion of the substratum surface for only two of the four PPFCs. Two models appeared to be flawed in the design of their inlets and outlets and allowed development of a stable velocity profile only for shear rates up to 0.5 and 500 s
−1
. For these PPFCs the inlet and outlet were curved, and the modeled shear rates deviated from the calculated shear rates by up to 75%. We concluded that PPFCs used for studies of microbial adhesion to surfaces should be designed so that their inlets and outlets are in line with the flow channel. Alternatively, the channel length should be increased to allow a greater length for the establishment of the desired hydrodynamic conditions.
Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the Human Subgingival Crevice
Authors: Ouverney Cleber C. , Armitage Gary C. , Relman David A.
Published: October 2003

Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells from healthy site samples.
Application of Host-Specific Bacteriophages to the Surface of Chicken Skin Leads to a Reduction in Recovery of
Campylobacter jejuni
Authors: Atterbury Robert J. , Connerton Phillippa L. , Dodd Christine E. R. , Rees Catherine E. D. , Connerton Ian F.
Published: October 2003

Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce
Campylobacter
contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable
Campylobacter jejuni
cells on artificially contaminated chicken skin.
Petroleum Pollution Bioremediation Using Water-Insoluble Uric Acid as the Nitrogen Source
Authors: Koren Omry , Knezevic Vishnia , Ron Eliora Z. , Rosenberg Eugene
Published: October 2003

The biodegradation of hydrocarbon pollutants in open systems is limited by the availability of a utilizable nitrogen source. This limitation can be overcome by using uric acid. Enrichment cultures grown on crude oil-uric acid media yielded mixed and pure cultures that degraded petroleum. In a simulated open system, uric acid bound to crude oil and was available for bacterial growth and petroleum biodegradation.
DpiA Binding to the Replication Origin ofEscherichia coliPlasmids and Chromosomes Destabilizes Plasmid Inheritance and Induces the Bacterial SOS Response
Authors: Miller Christine , Ingmer Hanne , Thomsen Line Elnif , Skarstad Kirsten , Cohen Stanley N.
Published: October 2003

ThedpiAanddpiBgenes ofEscherichia coli, which are orthologs of genes that regulate citrate uptake and utilization inKlebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in theK. pneumoniaepromoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response inE. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size—both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB—both of which act at A+T-rich replication origin sequences in theE. colichromosome and DpiA-targeted plasmids—reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.
Spontaneously Arising
mutL
Mutators in Evolving
Escherichia coli
Populations Are the Result of Changes in Repeat Length
Authors: Shaver Aaron C. , Sniegowski Paul D.
Published: October 2003

Over the course of thousands of generations of growth in a glucose-limited environment, 3 of 12 experimental populations of
Escherichia coli
spontaneously and independently evolved greatly increased mutation rates. In two of the populations, the mutations responsible for this increased mutation rate lie in the same region of the mismatch repair gene
mutL
. In this region, a 6-bp repeat is present in three copies in the gene of the wild-type ancestor of the experimental populations but is present in four copies in one of the experimental populations and two copies in the other. These in-frame mutations either add or delete the amino acid sequence LA in the MutL protein. We determined that the replacement of the wild-type sequence with either of these mutations was sufficient to increase the mutation rate of the wild-type strain to a level comparable to that of the mutator strains. Complementation of strains bearing the mutator mutations with wild-type copies of either
mutL
or the mismatch repair gene
uvrD
rescued the wild-type mutation rate. The position of the mutator mutations—in the region of MutL known as the ATP lid—suggests a possible deficiency in MutL's ATPase activity as the cause of the mutator phenotype. The similarity of the two mutator mutations (despite the independent evolutionary histories of the populations that gave rise to them) leads to a discussion of the potential adaptive role of DNA repeats.
Effects of Carboxy-Terminal Modifications and pH on Binding of a
Bacillus subtilis
Small, Acid-Soluble Spore Protein to DNA
Authors: Kosman Jeffrey , Setlow Peter
Published: October 2003

Variants of the wild-type
Bacillus subtilis
α/β-type small, acid-soluble spore protein (SASP) SspC
wt
were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA. SspC variants lacking one to three C-terminal residues were similar to SspC
wt
in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding. In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5- to 10-fold. C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC
ΔN11-D13K-C3
, for DNA was increased ≥20-fold over that of SspC
wt
. For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two- to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues. In vitro, the binding of SspC
ΔN11-D13K-C3
to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspC
wt
. However,
B. subtilis
spores lacking major α/β-type SASP and overexpressing SspC
ΔN11-D13K-C3
had a 10-fold-lower viability and far less UV and heat resistance than spores overexpressing SspC
wt
. This apparent lack of DNA protection by SspC
ΔN11-D13K-C3
in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspC
wt
, perhaps because of effects of SspC
ΔN11-D13K-C3
on gene expression in the forespore during sporulation. The latter results indicate that only moderately strong binding of α/β-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth.
Analysis of the Mobilization Region of the Broad-Host-Range IncQ-Like Plasmid pTC-F14 and Its Ability To Interact with a Related Plasmid, pTF-FC2
Authors: van Zyl Leonardo J. , Deane Shelly M. , Rawlings Douglas E.
Published: October 2003

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from
Acidithiobacillus caldus
that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five
mob
genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the
mobA
,
mobB
, and
mobC
genes, as well as the
oriT
gene. The
mobD
and
mobE
genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between
Escherichia coli
strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same
E. coli
host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2
mobD
and
mobE
gene products, the functions of which are still unknown. Mob protein interaction at the
oriT
regions was unidirectionally plasmid specific in that a plasmid with the
oriT
region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.
Changes in the Concentrations of Guanosine 5′-Diphosphate 3′-Diphosphate and the Initiating Nucleoside Triphosphate Account for Inhibition of rRNA Transcription in Fructose-1,6-Diphosphate Aldolase (
fda
) Mutants
Authors: Schneider David A. , Gourse Richard L.
Published: October 2003

Early screens for conditional lethal mutations that affected rRNA expression in
Escherichia coli
identified temperature-sensitive
fda
mutants (
fda
encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase). It was shown that these
fda
(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined. Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5′-diphosphate 3′-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of
fda
mutations on rRNA transcription.
Mutations Affecting DNA-Binding Activity of the MexR Repressor ofmexR-mexA-mexB-oprMOperon Expression
Authors: Saito Kohjiro , Akama Hiroyuki , Yoshihara Eisaku , Nakae Taiji
Published: October 2003

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bindmexOPDNA. Surprisingly, 20 mutations were located in the hydrophobic core region at α4, W1, α2, α3, and β2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.
The SKHR Motif Is Required for Biological Function of the VirR Response Regulator from
Clostridium perfringens
Authors: McGowan Sheena , O'Connor Jennifer R. , Cheung Jackie K. , Rood Julian I.
Published: October 2003

The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen
Clostridium perfringens
. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.
Nucleotide-Dependent Conformational Changes in the σ54-Dependent Activator DctD
Authors: Wang Ying-Kai , Park Sungdae , Nixon B. Tracy , Hoover Timothy R.
Published: October 2003

Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctDΔ1-142, a truncated and constitutively active form of the σ54-dependent activator DctD fromSinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of thedctApromoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctDΔ1-142during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2′(3′)-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctDΔ1-142and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.
Molecular Subtyping of
Vibrio cholerae
O1 and O139 by Pulsed-Field Gel Electrophoresis in Hong Kong: Correlation with Epidemiological Events from 1994 to 2002
Authors: Kam Kai Man , Luey Cindy Kit Yee , Tsang Yee Man , Law Choi Ping , Chu Man Yu , Cheung Tze Leung , Chiu Agatha Wai Huen
Published: October 2003

Two hundred twenty isolates of
Vibrio cholerae
O1 and O139 collected from 1994 to 2002 in Hong Kong were analyzed by pulsed-field gel electrophoresis (PFGE). Chromosomal DNAs from all
V. cholerae
isolates in agarose plugs were digested with the restriction enzyme
Not
I, resulting in 20 to 27 bands. Sixty distinctive PFGE patterns in the range of 10 to 300 kb were noted among 213 isolates typeable by PFGE. By comparing the common PFGE patterns obtained from four well-defined outbreaks of
V. cholerae
O1 and O139 with those obtained from other, epidemiologically unrelated isolates during the study period, indistinguishable and similar PFGE patterns were identified, indicating their close relatedness, in agreement with the results of epidemiological investigations. Heterogeneous PFGE patterns (with four to six banding differences), however, were identified among strains that were imported from other parts of Asia, including Indonesia, India, and Pakistan. Correlations with epidemiological information further support the usefulness of PFGE as an epidemiological tool in laboratory investigations of suspected outbreaks. Standardization of PFGE methodology will allow international comparison of fingerprint patterns and will form the basis of a laboratory network for tracking
V. cholerae
.
Evaluation of Reverse Transcription-PCR Assays for Rapid Diagnosis of Severe Acute Respiratory Syndrome Associated with a Novel Coronavirus
Authors: Yam W. C. , Chan K. H. , Poon L. L. M. , Guan Y. , Yuen K. Y. , Seto W. H. , Peiris J. S. M.
Published: October 2003

The reverse transcription (RT)-PCR protocols of two World Health Organization (WHO) severe acute respiratory syndrome (SARS) network laboratories (WHO SARS network laboratories at The University of Hong Kong [WHO-HKU] and at the Bernhard-Nocht Institute in Hamburg, Germany [WHO-Hamburg]) were evaluated for rapid diagnosis of a novel coronavirus (CoV) associated with SARS in Hong Kong. A total of 303 clinical specimens were collected from 163 patients suspected to have SARS. The end point of both WHO-HKU and WHO-Hamburg RT-PCR assays was determined to be 0.1 50% tissue culture infective dose. Using seroconversion to CoV as the “gold standard” for SARS CoV diagnosis, WHO-HKU and WHO-Hamburg RT-PCR assays exhibited diagnostic sensitivities of 61 and 68% (nasopharyngeal aspirate specimens), 65 and 72% (throat swab specimens), 50 and 54% (urine specimens), and 58 and 63% (stool specimens), respectively, with an overall specificity of 100%. For patients confirmed to have SARS CoV and from whom two or more respiratory specimens were collected, testing the second specimen increased the sensitivity from 64 and 71% to 75 and 79% for the WHO-HKU and WHO-Hamburg RT-PCR assays, respectively. Testing more than one respiratory specimen will maximize the sensitivity of PCR assays for SARS CoV.
New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Palmer Sarah , Wiegand Ann P. , Maldarelli Frank , Bazmi Holly , Mican JoAnn M. , Polis Michael , Dewar Robin L. , Planta Angeline , Liu Shuying , Metcalf Julia A. , Mellors John W. , Coffin John M.
Published: October 2003

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10
6
per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.
Madurella mycetomatisStrains from Mycetoma Lesions in Sudanese Patients Are Clonal
Authors: Ahmed Abdalla , van de Sande Wendy , Verbrugh Henri , Fahal Ahmed , van Belkum Alex
Published: October 2003

Molecular diversity among clinical isolates ofMadurella mycetomatis, the prime fungal agent of human mycetoma in Sudan, could possibly explain the diverse clinical presentations of this severely debilitating infectious disease. In addition, culture-independent DNA-mediated typing tests need to be developed for this organism, sinceM. mycetomatisDNA, but not the organism itself, can be identified in soil, the material from which infections are thought to originate. A collection of 38 different clinicalM. mycetomatisisolates was characterized by large-scale random amplification of polymorphic DNA using 20 different primer species. These analyses, involving at least 2,600 annealing sites, showed a complete lack of DNA fingerprint variation among the various isolates. From the resulting homogeneous DNA fingerprints, seven fragments were cloned and sequenced, and novel, species-specific PCR restriction fragment length polymorphism (RFLP) tests were designed. The seven PCR RFLP tests were successfully performed on the 38 differentM. mycetomatisstrains. However, again allM. mycetomatisDNA patterns obtained appeared to be identical, whereas patterns produced using DNAs from other fungal species were clearly discriminatory. These results suggest that there is little genetic variation among clinically relevantM. mycetomatisstrains from Sudan. The data tentatively imply that different manifestations of mycetoma are due to differences in host susceptibility rather than differential virulence of the causative agent.
Clonal Characterization of
Staphylococcus aureus
by Multilocus Restriction Fragment Typing, a Rapid Screening Approach for Molecular Epidemiology
Authors: Diep Binh An , Perdreau-Remington Françoise , Sensabaugh George F.
Published: October 2003

We have developed a rapid and simplified approach for the strain characterization of
Staphylococcus aureus
on the basis of multilocus sequence typing (MLST) in which sequence variations in the MLST housekeeping gene loci are detected by restriction fragment pattern analysis rather than sequencing; we refer to this approach as multilocus restriction fragment typing (MLRFT). Briefly, MLRFT for
S. aureus
involves the PCR amplification of each of the seven MLST housekeeping gene loci by using the same primer pairs used in MLST. The amplicons are then digested directly with one or two restriction enzymes and the restriction fragments are resolved by agarose gel electrophoresis. Projection from published MLST data shows that MLRFT captures about 95% of the genetic diversity detected by MLST. The MLRFT approach was validated with a set of 59 methicillin-susceptible and 44 methicillin-resistant
S. aureus
isolates from community-acquired and nosocomial sources which had previously been characterized by pulsed-field gel electrophoresis (PFGE). MLRFT resolved the 103 isolates into 15 restriction fragment types, giving a discrimination index of 89.0%. Clonal groupings established by MLRFT correlated well with those established by PFGE. In short, MLRFT provides a convenient alternative to MLST and PFGE because it requires minimal laboratory facilities and is relatively simple and inexpensive to perform.
Evaluation of Protocol Using Gene Capture and PCR for Detection of
Helicobacter pylori
DNA in Feces
Authors: MacKay W. G. , Williams C. L. , McMillan M. , Ndip R. N. , Shepherd A. J. , Weaver L. T.
Published: October 2003

The route of transmission of
Helicobacter pylori
, which is usually acquired in childhood and is one of the most common bacterial infections in humans, remains undetermined. Mapping the distribution of
H
.
pylori
genotypes within families could help to determine the routes of transmission and risk factors. Here we describe a noninvasive method for obtaining
H
.
pylori
DNA isolates from the feces of children. Children presenting with gastrointestinal symptoms at the Royal Hospital for Sick Children were tested for gastric
H
.
pylori
colonization by using the
13
C-urea breath test (UBT) and were asked to provide fecal samples, which were tested for
H
.
pylori
by using the HpSA fecal antigen test. DNA was purified from fecal samples by using a novel method of gene capture with subsequent
H
.
pylori
PCR analysis. Fifteen UBT-positive and 15 UBT-negative children participated in the study. The positive and negative predictive values for the assay were 80 and 100%, respectively. Fecal DNA purification followed by
H
.
pylori
PCR analysis is an effective tool for harvesting
H
.
pylori
DNA isolates from the feces of children. This technique may be developed to allow the diagnosis and noninvasive genotyping of
H
.
pylori
in children and their families.