bluuebunny/crossref_metadata_2025 huggingface dataset:
In Silico Model as a Tool for Interpretation of Intestinal Infection Studies
Authors: de Jong Peter , Vissers Marc M. M. , van der Meer Roelof , Bovee-Oudenhoven Ingeborg M. J.
Published: November 2006

In nutrition research the number of human in vivo experiments is limited because of the many restrictions and the high costs of testing in humans. Up to now predictive computer models aiming to enhance research have been rare or too complex, with many nonmeasurable adjustable parameters. This study aimed to develop a basic physicochemical computer model for a first quantitative interpretation of results obtained from in vivo intestinal experiments with bacteria. This new modeling approach is validated with results obtained from gut infection studies in vivo. The design of the model is described, and its ability to reproduce experimental data is evaluated. The model predictions are compared with new experimental data. The phenomena that take place in the gastrointestinal tract are summarized by model constants for growth, adherence, and release of bacteria. Although the model is far from describing all details and many processes in the intestine are combined, the model calculation results lead to reasonable conclusions and interesting hypotheses. One of these hypotheses concluded from the model outcomes is that
Escherichia coli
bacteria have a much lower intestinal growth rate in humans than in rats. Extra laboratory validation experiments proved the reliability of this hypothesis predicted by the model. In addition, the known protective effect of dietary calcium and detrimental effect of clindamycin on the growth and adherence of
Salmonella
bacteria could be quantified. From these results it is clear that the model enhances the interpretation of in vivo gastrointestinal experiments and will facilitate research trajectories towards new functional foods that improve resistance to pathogenic bacteria in humans.
Vertical Transmission of Diverse Microbes in the Tropical SpongeCorticiumsp
Authors: Sharp Koty H. , Eam Boreth , Faulkner D. John , Haygood Margo G.
Published: November 2006

Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical spongeCorticiumsp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.
Operon Prediction for Sequenced Bacterial Genomes without Experimental Information
Authors: Bergman Nicholas H. , Passalacqua Karla D. , Hanna Philip C. , Qin Zhaohui S.
Published: November 2006

Various computational approaches have been proposed for operon prediction, but most algorithms rely on experimental or functional data that are only available for a small subset of sequenced genomes. In this study, we explored the possibility of using phylogenetic information to aid in operon prediction, and we constructed a Bayesian hidden Markov model that incorporates comparative genomic data with traditional predictors, such as intergenic distances. The prediction algorithm performs as well as the best previously reported method, with several significant advantages. It uses fewer data sources and so it is easier to implement, and the method is more broadly applicable than previous methods—it can be applied to essentially every gene in any sequenced bacterial genome. Furthermore, we show that near-optimal performance is easily reached with a generic set of comparative genomes and does not depend on a specific relationship between the subject genome and the comparative set. We applied the algorithm to the
Bacillus anthracis
genome and found that it successfully predicted all previously verified
B. anthracis
operons. To further test its performance, we chose a predicted operon (BA1489-92) containing several genes with little apparent functional relatedness and tested their cotranscriptional nature. Experimental evidence shows that these genes are cotranscribed, and the data have interesting implications for
B. anthracis
biology. Overall, our findings show that this algorithm is capable of highly sensitive and accurate operon prediction in a wide range of bacterial genomes and that these predictions can lead to the rapid discovery of new functional relationships among genes.
Prevalence of ColE1-Like Plasmids and Colicin K Production among Uropathogenic
Escherichia coli
Strains and Quantification of Inhibitory Activity of Colicin K
Authors: Rijavec Matija , Budič Maruška , Mrak Peter , Müller-Premru Manica , Podlesek Zdravko , Žgur-Bertok Darja
Published: November 2006

Colicin K exhibited pronounced inhibitory activity against uropathogenic
Escherichia coli
(UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS
2
.
Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts
Authors: Partida-Martinez Laila P. , Flores de Looß Carina , Ishida Keishi , Ishida Mie , Roth Martin , Buder Katrin , Hertweck Christian
Published: November 2006

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal
Rhizopus microsporus
strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus
Burkholderia
. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since
Rhizopus microsporus
and related
Rhizopus
species are frequently used in food preparations such as tempeh and sufu.
Exopolysaccharide Expression in
Lactococcus lactis
subsp.
cremoris
Ropy352: Evidence for Novel Gene Organization
Authors: Knoshaug Eric P. , Ahlgren Jeff A. , Trempy Janine E.
Published: November 2006

Lactococcus lactis
subsp.
cremoris
Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain,
L. lactis
subsp.
cremoris
EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of
L. lactis
subsp.
cremoris
Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy
L. lactis
subsp.
cremoris
EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames,
epsM
and
epsN
, encoding novel glycosyltransferases bordered by IS
S1
elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS
981
and IS
982
elements, and an independent gene,
epsU
. Two and possibly three of these regions are involved in
L. lactis
subsp.
cremoris
Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in
Lactococcus
.
Characterization of Chimeric
Bacillus thuringiensis
Vip3 Toxins
Authors: Fang Jun , Xu Xiaoli , Wang Ping , Zhao Jian-Zhou , Shelton Anthony M. , Cheng Jiaan , Feng Ming-Guang , Shen Zhicheng
Published: November 2006

Bacillus thuringiensis
vegetative insecticidal proteins (Vip) are potential alternatives for
B. thuringiensis
endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of
B. thuringiensis
isolates resulted in the cloning of
vip3Ac1
. Vip3Ac1 showed high insecticidal activity against the fall armyworm
Spodoptera frugiperda
and the cotton bollworm
Helicoverpa zea
but very low activity against the silkworm
Bombyx mori
. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer
Ostrinia nubilalis
, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (
Trichoplusia ni
) that is highly resistant to the
B. thuringiensis
endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between
B. thuringiensis
Cry1A proteins and Vip3A toxins. The results in this study demonstrated that
vip3Ac1
and its chimeric
vip3
genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.
Preparation of Monoclonal Antibodies for Detection and Identification of
Francisella tularensis
Authors: Hotta Akitoyo , Uda Akihiko , Fujita Osamu , Tanabayashi Kiyoshi , Yamada Akio
Published: November 2006

Monoclonal antibodies (MAbs) against
Francisella tularensis
were obtained. Three MAbs specifically reacted with
F. tularensis
, while four MAbs reacted with other members of the genus
Francisella
as well. Fluorescent isothiocyanate-conjugated MAbs unequivocally stained bacterial cells in specimens from experimentally infected mice. Two MAbs agglutinated
F. tularensis
antigen in the agglutination tests. These MAbs should improve methods for detection and identification of
F. tularensis
.
Limited Functional Redundancy and Oscillation of Cyclins in Multinucleated
Ashbya gossypii
Fungal Cells
Authors: Hungerbuehler A. Katrin , Philippsen Peter , Gladfelter Amy S.
Published: November 2006

Cyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in
A. gossypii
, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in
A. gossypii
to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in
A. gossypii
are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells.
Phosphoinositides Are Involved in Control of the Glucose-Dependent Growth Resumption That Follows the Transition Phase inStreptomyces lividans
Authors: Chouayekh H. , Nothaft H. , Delaunay S. , Linder M. , Payrastre B. , Seghezzi N. , Titgemeyer F. , Virolle M. J.
Published: November 2006

The interruption of thesblAgene ofStreptomyces lividanswas previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed α-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of thesblAmutant strain.sblAwas shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression ofsblAthat takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control ofsblAexpression was shown to depend on two operator sites. One, located just upstream of the −35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTGsblAtranslational start codon, may be involved in the regulation of the degradation of thesblAtranscript. This study suggests that phosphoinositides constitute important regulatory molecules inStreptomyces, as they do in eukaryotes.
Crystal Structure of Uroporphyrinogen Decarboxylase fromBacillus subtilis
Authors: Fan Jun , Liu Qun , Hao Quan , Teng Maikun , Niu Liwen
Published: November 2006

Uroporphyrinogen decarboxylase (UROD) is a branch point enzyme in the biosynthesis of the tetrapyrroles. It catalyzes the decarboxylation of four acetate groups of uroporphyrinogen III to yield coproporphyrinogen III, leading to heme and chlorophyll biosynthesis. UROD is a special type of nonoxidative decarboxylase, since no cofactor is essential for catalysis. In this work, the first crystal structure of a bacterial UROD,Bacillus subtilisUROD (URODBs), has been determined at a 2.3 Å resolution. The biological unit of URODBswas determined by dynamic light scattering measurements to be a homodimer in solution. There are four molecules in the crystallographic asymmetric unit, corresponding to two homodimers. Structural comparison of URODBswith eukaryotic URODs reveals a variation of two loops, which possibly affect the binding of substrates and release of products. Structural comparison with the human UROD-coproporphyrinogen III complex discloses a similar active cleft, with five invariant polar residues (Arg29, Arg33, Asp78, Tyr154, and His322) and three invariant hydrophobic residues (Ile79, Phe144, and Phe207), in URODBs. Among them, Asp78 may interact with the pyrrole NH groups of the substrate, and Arg29 is a candidate for positioning the acetate groups of the substrate. Both residues may also play catalytic roles.
Identification of Genes Regulated by the
cepIR
Quorum-Sensing System in
Burkholderia cenocepacia
by High-Throughput Screening of a Random Promoter Library
Authors: Subsin Benchamas , Chambers Catherine E. , Visser Michelle B. , Sokol Pamela A.
Published: November 2006

The
Burkholderia cenocepacia cepIR
quorum-sensing system regulates expression of extracellular proteases, chitinase, and genes involved in ornibactin biosynthesis, biofilm formation, and motility. In a genome-wide screen we identified
cepIR
-regulated genes by screening a random promoter library of
B. cenocepacia
K56-2 constructed in a luminescence reporter detection plasmid for differential expression in response to
N
-octanoyl-
l
-homoserine lactone (OHL). Eighty-nine clones were identified; in 58 of these clones expression was positively regulated by
cepIR
, and in 31 expression was negatively regulated by
cepIR
. The expression profiles of the 89 promoter clones were compared in the
cepI
mutant K56-dI2 in medium supplemented with 30 pM OHL and K56-2 to confirm that the presence of OHL restored expression to wild-type levels. To validate the promoter library observations and to determine the effect of a
cepR
mutation on expression of selected genes, the mRNA levels of nine genes whose promoters were predicted to be regulated by
cepR
were quantitated by quantitative reverse transcription-PCR in the wild type and
cepI
and
cepR
mutants. The expression levels of all nine genes were similar in the
cepI
and
cepR
mutants and consistent with the promoter-
lux
reporter activity. The expression of four selected
cepIR
-regulated gene promoters was examined in a
cciIR
mutant, and two of these promoters were also regulated by
cciIR
. This study extends our understanding of genes whose expression is influenced by
cepIR
and indicates the global regulatory effect of the
cepIR
system in
B. cenocepacia
.
Analysis of Storage Lipid Accumulation in
Alcanivorax borkumensis
: Evidence for Alternative Triacylglycerol Biosynthesis Routes in Bacteria
Authors: Kalscheuer Rainer , Stöveken Tim , Malkus Ursula , Reichelt Rudolf , Golyshin Peter N. , Sabirova Julia S. , Ferrer Manuel , Timmis Kenneth N. , Steinbüchel Alexander
Published: November 2006

Marine hydrocarbonoclastic bacteria, like
Alcanivorax borkumensis
, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in
A. borkumensis
strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in
atfA1
and
atfA2
knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of
A. borkumensis
were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.
Genome-Wide Screening of Genes Required for Swarming Motility inEscherichia coliK-12
Authors: Inoue Tetsuyoshi , Shingaki Ryuji , Hirose Shotaro , Waki Kaori , Mori Hirotada , Fukui Kazuhiro
Published: November 2006

Escherichia coliK-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disruptedE. coliK-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in theE. coliK-12 strain.
Involvement of the
cynABDS
Operon and the CO
2
-Concentrating Mechanism in the Light-Dependent Transport and Metabolism of Cyanate by Cyanobacteria
Authors: Espie George S. , Jalali Farid , Tong Tommy , Zacal Natalie J. , So Anthony K.-C.
Published: November 2006

The cyanobacteria
Synechococcus elongatus
strain PCC7942 and
Synechococcus
sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO
−
) to CO
2
and NH
3
through the action of a cytosolic cyanase which required HCO
3
−
as a second substrate. The ability to metabolize NCO
−
relied on three essential elements: proteins encoded by the
cynABDS
operon, the biophysical activity of the CO
2
-concentrating mechanism (CCM), and light. Inactivation of
cynS
, encoding cyanase, and
cynA
yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO
2
, HCO
3
−
, NO
3
−
, NO
2
−
, Cl
−
, PO
4
2−
, and SO
4
2−
did not contribute to the cellular flux of NCO
−
and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO
−
transporter. In the
S. elongatus
strain PCC7942 Δ
chpX
Δ
chpY
mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (C
i
) (HCO
3
−
+ CO
2
) pool. Unlike wild-type cells, the rate of NCO
−
decomposition by the Δ
chpX
Δ
chpY
mutant was severely depressed at low external C
i
concentrations, indicating that the CCM was essential in providing HCO
3
−
for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO
−
and C
i
. Putative
cynABDS
operons were identified in the genomes of diverse
Proteobacteria
, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.
The Inner Cavity ofEscherichia coliDegP Protein Is Not Essentialfor Molecular Chaperone and Proteolytic Activity
Authors: Jomaa Ahmad , Damjanovic Daniela , Leong Vivian , Ghirlando Rodolfo , Iwanczyk Jack , Ortega Joaquin
Published: November 2006

TheEscherichia coliDegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28°C, but efficiently degrading unfolded proteins above 28°C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.
Regulation of Denitrification Genes in
Neisseria meningitidis
by Nitric Oxide and the Repressor NsrR
Authors: Rock Jonathan D. , Thomson Melanie J. , Read Robert C. , Moir James W. B.
Published: November 2006

The human pathogen
Neisseria meningitidis
is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by
aniA
) and nitric oxide (NO) reductase (encoded by
norB
). Here, we show that in
N. meningitidis
MC58
norB
is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but
norB
expression is derepressed by NO in an NsrR-dependent manner.
nsrR
-deficient mutants grow by denitrification more rapidly than wild-type
N. meningitidis
, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of
aniA
(unlike that of
norB
) is not lifted in the presence of NO. The role of NsrR in the control of expression of
aniA
is linked to the function of the anaerobic activator protein FNR: analysis of
nsrR
and
fnr
single and
nsrR fnr
double mutants carrying an
aniA
promoter
lacZ
fusion indicates that the role of NsrR is to prevent FNR-dependent
aniA
expression under aerobic conditions, indicating that FNR in
N. meningitidis
retains considerable activity aerobically.
Membrane-Spanning and Periplasmic Segments of CcmI Have Distinct Functions during Cytochrome
c
Biogenesis in
Rhodobacter capsulatus
Authors: Sanders Carsten , Boulay Clémence , Daldal Fevzi
Published: November 2006

In gram-negative bacteria, like
Rhodobacter capsulatus
, about 10 membrane-bound components (CcmABCDEFGHI and CcdA) are required for periplasmic maturation of
c
-type cytochromes. These components perform the chaperoning and thio-oxidoreduction of the apoproteins as well as the delivery and ligation of the heme cofactors. In the absence of any of these components, including CcmI, proposed to act as an apocytochrome
c
chaperone,
R. capsulatus
does not have the ability to produce holocytochromes
c
or consequently to exhibit photosynthetic growth and cytochrome
cbb
3
oxidase activity. Previously, we have demonstrated that null mutants of CcmI partially overcome cytochrome
c
deficiency phenotypes upon overproduction of the CcmF-
R. capsulatus
CcmH (CcmF-CcmH
Rc
) couple in a growth medium-dependent manner and fully bypass these defects by additional overproduction of CcmG. Here, we show that overproduction of the CcmF-CcmH
Rc
couple and overproduction of the N-terminal membrane-spanning segment of CcmI (CcmI-1) have similar suppression effects of cytochrome
c
maturation defects in CcmI-null mutants. Likewise, additional overproduction of CcmG, the C-terminal periplasmic segment of CcmI (CcmI-2), or even of apocytochrome
c
2
also provides complementation abilities similar to those of these mutants. These results indicate that the two segments of CcmI have different functions and support our earlier findings that two independent steps are required for full recovery of the loss of CcmI function. We therefore propose that CcmI-1 is part of the CcmF-CcmH
Rc
-dependent heme ligation, while CcmI-2 is involved in the CcdA- and CcmG-dependent apoprotein thioreduction steps, which intersect at the level of CcmI during cytochrome
c
biogenesis.
Structural Characterization of a Specific Glycopeptidolipid Containing a Novel
N
-Acyl-Deoxy Sugar from
Mycobacterium intracellulare
Serotype 7 and Genetic Analysis of Its Glycosylation Pathway
Authors: Fujiwara Nagatoshi , Nakata Noboru , Maeda Shinji , Naka Takashi , Doe Matsumi , Yano Ikuya , Kobayashi Kazuo
Published: November 2006

The nontuberculous
Mycobacterium avium-Mycobacterium intracellulare
complex (MAC) is distributed ubiquitously in the environment and is an important cause of respiratory and lymphatic disease in humans and animals. These species produce polar glycopeptidolipids (GPLs), and of particular interest is their serotype-specific antigenicity. Structurally, GPLs contain an
N
-acylated tetrapeptide-amino alcohol core that is glycosylated at the C terminal with 3,4-di-
O
-methyl rhamnose and at the
d
-
allo
-threonine with a 6-deoxy-talose. This serotype nonspecific GPL is found in all MAC species. The serotype-specific GPLs are further glycosylated with a variable haptenic oligosaccharide at 6-deoxy-talose. At present, 31 distinct serotype-specific GPLs have been identified on the basis of oligosaccharide composition, and the complete structures of 14 serotype-specific GPLs have been defined. It is considered that the modification of the GPL structure plays an important role in bacterial physiology, pathogenesis, and host immune responses. In this study, we defined the complete structure of a novel serotype 7 GPL that has a unique terminal amido sugar. The main molecular mass is 1,874, and attached to the tetrapeptide-amino alcohol core is the serotype 7-specific oligosaccharide unit of 4-2′-hydroxypropanoyl-amido-4,6-dideoxy-2-
O
-methyl-β-hexose-(1→3)-α-
l
-rhamnose-(1→3)-α-
l
-rhamnose-(1→3)-α-
l
-rhamnose-(1→2)-α-
l
-6-deoxy-talose. Moreover, we isolated and characterized the serotype 7-specific gene cluster involved in glycosylation of the oligosaccharide. Nine open reading frames (ORFs) were observed in the cluster. Based on the sequence homology, the ORFs are thought to participate in the biosynthesis of the serotype 7 GPL.
Prospective Evaluation of the Hybrid Capture 2 and AMPLICOR Human Papillomavirus (HPV) Tests for Detection of 13 High-Risk HPV Genotypes in Atypical Squamous Cells of Uncertain Significance
Authors: Halfon Philippe , Trepo Elisabeth , Antoniotti Gilles , Bernot Catherine , Cart-Lamy Philippe , Khiri Hacène , Thibaud Didier , Marron Jean , Martineau Agnès , Pénaranda Guillaume , Benmoura Dominique , Blanc Bernard
Published: November 2006

The use of high-risk human papillomavirus (hrHPV) testing as an adjunct to cervical cytology in population-based screening programs is currently based on DNA hybridization and PCR assays. The aim of this study was to prospectively assess the diagnostic performance of the Hybrid Capture 2 test (HC2; Digene Corporation) in comparison with that of the recently developed PCR-based AMPLICOR HPV test (Roche Molecular Systems) for the detection of 13 hrHPV types. A reverse line blot hybridization assay (Innogenetics) was used as an internal reference standard in discordant cases. Two hundred seventy-one patients with atypical squamous cells of uncertain significance (ASCUS) in cervical samples underwent hrHPV testing. The chi-square test was performed to compare respective proportions. Totals of 160/271 (59%) and 156/271 (58%) were found to be positive for hrHPV with HC2 and AMPLICOR, respectively. Concordant results were obtained for 235 (86.7%) of the 271 samples (kappa statistic, 0.73 ± 0.04). Considering types 26, 53, and 66 as oncogenic types, negative predictive values (NPVs) of HC2 and AMPLICOR were 92.8% and 87.8%, respectively (difference was not significant), and their respective accuracies were 94.8% and 91.9% (difference was not significant). Considering types 26, 53, and 66 as not oncogenic, the respective HC2 and AMPLICOR NPVs were 92.8% and 97.4% (difference was not significant), and accuracy was significantly higher for the AMPLICOR assay (95.9% versus 90.8% for HC2) (
P
< 0.05). For ASCUS samples, the NPV was 92.8% for HC2 testing and might be compromised if the copy number of HPV DNA was low. The NPV was 97.4% for the AMPLICOR assay and might be compromised if HPV types 26, 53, and 66 were considered oncogenic. The accuracy of these two assays is good and is compatible with routine clinical use in the triage of ASCUS cases.
Antibiotic-Resistant Invasive Pneumococcal Clones in Italy
Authors: Gherardi Giovanni , Fallico Loredana , Del Grosso Maria , Bonanni Federica , D'Ambrosio Fabio , Manganelli Riccardo , Palù Giorgio , Dicuonzo Giordano , Pantosti Annalisa
Published: November 2006

A total of 105 multiple-antibiotic-resistant invasive pneumococcal isolates recovered in Italy from 2001 to 2003 were genetically characterized. Of these, 40 were penicillin-nonsusceptible (PNSSP) and 65 were penicillin-susceptible (PSSP)
Streptococcus pneumoniae
strains. Among the PNSSP isolates, 8 and 11 different restriction profiles were obtained for the
pbp2b
and
pbp2x
genes, respectively. Clonal groups were established on the basis of analysis of both pulsed-field gel electrophoresis (PFGE) types and multilocus sequence typing (MLST). Several international clones, such as Spain
23F
-1/ST81, Spain
6B
-2/ST90, Spain
9V
-3/ST156, and Sweden
15A
-25/ST263, were identified among the PNSSP isolates. Other, smaller clones, such as the minor Spanish 19F clone/ST88 and Denmark
14
-32/ST230, were also found. Among the PSSP isolates, clones related to England
14
-9/ST9, Greece
6B
-22/ST273, and Portugal
19F
-21/ST177 were found. In addition, two large clones comprised nonvaccine serotypes. One, comprising serotype 3 isolates, corresponded to the clone Netherlands
3
-31/ST180; the other, comprising serotype 15B/C isolates, ST474, was not related to any previously described clone. Two small clusters related to the newly described clones Greece
21
-30/ST193 and Netherlands
15B
-37/ST199 included isolates with unrelated PFGE profiles. An unusual finding was the inability to obtain the MLST allelic profile for an isolate of serotype 19A, belonging to the Sweden
15A
-25/ST263 clone, due to a large deletion of the
xpt
gene. Capsular switching was observed among both PNSSP and PSSP isolates and involved also serotypes not included in the 7-valent pneumococcal conjugate vaccine (PCV7), such as serotypes 15B/C and 19A. Since antibiotic-resistant nonvaccine serotype clones are present in Italy, continuous monitoring of pneumococcal epidemiology should be carried out in the PCV7 era.
Evidence for a Link between Parasite Genotype and Outcome of Infection with
Entamoeba histolytica
Authors: Ali Ibne Karim M. , Mondal Utpal , Roy Shantanu , Haque Rashidul , Petri William A. , Clark C. Graham
Published: November 2006

The factors determining whether a person infected with
Entamoeba histolytica
develops disease remain obscure. To investigate whether the parasite genome contributes to the outcome, we have investigated the distribution of parasite genotypes among
E. histolytica
-infected individuals in Bangladesh. Samples were obtained from individuals who either were asymptomatic, had diarrhea/dysentery, or had developed a liver abscess. Genotypes were determined by using six tRNA-linked polymorphic markers, and their distributions among the three sample groups were evaluated. A significant population differentiation in the genotype distribution was found for four of the six individual markers as well as for the combined genotypes, suggesting that the parasite genome does contribute in some way to the outcome of infection with
E. histolytica
. The markers themselves do not indicate the nature of the underlying genetic differences, but they may be linked to loci that do have an impact on the outcome of infection.
PCR-Based Diagnosis of
Naegleria
sp. Infection in Formalin-Fixed and Paraffin-Embedded Brain Sections
Authors: Schild Marc , Gianinazzi Christian , Gottstein Bruno , Müller Norbert
Published: November 2006

We developed a real-time PCR which allowed the highly sensitive detection of
Naegleria fowleri
in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.
A Multiplex Ligase Detection Reaction-Fluorescent Microsphere Assay for Simultaneous Detection of Single Nucleotide Polymorphisms Associated with
Plasmodium falciparum
Drug Resistance
Authors: Carnevale Eric P. , Kouri Drew , DaRe Jeana T. , McNamara David T. , Mueller Ivo , Zimmerman Peter A.
Published: November 2006

Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of
Plasmodium falciparum
drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the
P. falciparum dhfr
(9 alleles),
dhps
(10 alleles), and
pfcrt
(3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were
P. falciparum
negative and 453 samples were
P. falciparum
positive by a
Plasmodium
species assay and all three gene assays (concordance, 82.2%). For
P. falciparum
infections where the assay for each gene was positive, 2 samples carried resistance alleles for all three genes, 299 carried resistance alleles for
dhfr
and
pfcrt
, 131 carried resistance alleles for only one gene (
dhfr
[
n
= 40],
dhps
[
n
= 1], or
pfcrt
[
n
= 90]), and 21 carried only sensitive alleles at all three genes. Mixed-strain infections characterized 100 samples. Overall, 95.4% (432/453) of
P. falciparum
-infected samples carried at least one allele associated with resistance to Fansidar or chloroquine. In view of the fact that 86.3% (391/453) of
P. falciparum
-infected samples carried
pfcrt
mutations, chloroquine is largely ineffective against
P. falciparum
in Papua New Guinea. Surveillance of additional
dhfr
and
dhps
polymorphisms in order to monitor the continued effectiveness of Fansidar is recommended.
Controlled Clinical Comparison of VersaTREK and BacT/ALERT Blood Culture Systems
Authors: Mirrett Stanley , Hanson Kimberly E. , Reller L. Barth
Published: November 2006

To assess the relative yields in automated microbial detection systems of bacteria and yeasts isolated from the blood of adult patients with suspected sepsis, we compared the new VersaTREK system (VTI) (TREK Diagnostic Systems, Cleveland, OH) to the BacT/ALERT 3D system (3D) (bioMérieux, Inc., Durham, NC). Identical protocols were followed for the two systems. Paired aerobic (REDOX 1) and anaerobic (REDOX 2) VTI media were compared with standard aerobic (SA) and anaerobic (SN) 3D media; each of the four culture bottles was filled with 6 to 9 ml of blood. All bottles flagged positive by the instruments were subcultured to determine both true-positive (growth) and false-positive (no growth) cultures. Additionally, to assess false-negative bottles, terminal subcultures were done on all negative companion bottles to true-positive bottles. All isolates were identified by standard methods. All 4 bottles were adequately filled and yielded 413 clinically significant isolates in 5,389 (79%) of the 6,786 4-bottle sets obtained. Although no overall difference in yield or in time to detection was detected between the two systems, significantly more streptococci and enterococci as a group were detected by VTI. Moreover, significantly more microorganisms were detected by VTI for patients receiving antimicrobial therapy. The two systems were comparable (
P
, not significant) at detecting the 179 unimicrobial episodes of bacteremia seen. False-positive rates for aerobic and anaerobic bottles, respectively, were 1.6% and 0.9% for VTI and 0.7% and 0.8% for 3D. We conclude that the VTI and 3D systems are comparable for detection of bloodstream infections with bacteria or yeasts.
Isolation and Antimicrobial Susceptibilities of Chlamydial Isolates from Western Barred Bandicoots
Authors: Kumar Swati , Kutlin Andrei , Roblin Patricia , Kohlhoff Stephan , Bodetti Tracey , Timms Peter , Hammerschlag Margaret R.
Published: November 2006

A range of species of
Chlamydiales
have previously been detected in a variety of Australian marsupials, including koalas and western barred bandicoots. Thirty-seven ocular, urogenital, or nasal swabs were obtained from 21 wild western barred bandicoots.
Chlamydia
culture and antibiotic susceptibility testing were performed for cycloheximide-treated HEp-2 cells in 96-well microtiter plates.
Chlamydia
spp. were isolated from 11 specimens from 9 (42.8%) bandicoots. All isolates were identified as
Chlamydiales
by conventional PCR with 16S and 23S rRNA gene primers specific to
Chlamydiales
and were confirmed to be
Chlamydia pneumoniae
by a
C. pneumoniae
-specific
ompA
-based real-time PCR assay and 16S rRNA and 23S rRNA gene signature sequence analyses. The MICs of azithromycin, doxycycline, ciprofloxacin, and enrofloxacin for 10
C. pneumoniae
isolates from these bandicoots ranged from 0.015 to 1 μg/ml, 0.25 to 1 μg/ml, 0.25 to 2 μg/ml, and 0.25 to 0.5 μg/ml, respectively. The MICs at which 90% of isolates were inhibited and the minimal bactericidal concentrations were within the ranges reported previously for human isolates of
C. pneumoniae.
Globicatella sanguinis
Is an Etiological Agent of Ventriculoperitoneal Shunt-Associated Meningitis
Authors: Seegmüller I. , van der Linden M. , Heeg C. , Reinert R. R.
Published: November 2006

Globicatella sanguinis
is a very rare isolate in clinical samples. We present a case of meningitis in a 69-year-old female patient after implantation of an external left ventricular drainage due to a hydrocephalus. She recovered after antibiotic treatment with ceftriaxone.
Catabacter hongkongensisgen. nov., sp. nov., Isolated from Blood Cultures of Patients from Hong Kong and Canada
Authors: Lau Susanna K. P. , McNabb Alan , Woo Gibson K. S. , Hoang Linda , Fung Ami M. Y. , Chung Liliane M. W. , Woo Patrick C. Y. , Yuen Kwok-Yung
Published: November 2006

Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37°C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences fromClostridium propionicum,Clostridium neopropionicum, andAtopobium minutum, respectively. The G+C content of strain HKU16Tis 40.2% ± 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species,Catabacter hongkongensisgen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family,Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility ofC. hongkongensis.Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.
Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens
Authors: Lau Anna , Chen Sharon , Sorrell Tania , Carter Dee , Malik Richard , Martin Patricia , Halliday Catriona
Published: November 2006

Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n= 38) or solely histologically proven (n= 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species ofCandida,Cryptococcus,Trichosporon,Aspergillus,Fusarium,Scedosporium,Exophiala,Exserohilum,Apophysomyces,Actinomucor, andRhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n= 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.
Mucor circinelloides
Was Identified by Molecular Methods as a Cause of Primary Cutaneous Zygomycosis
Authors: Iwen Peter C. , Sigler Lynne , Noel Rhonda K. , Freifeld Alison G.
Published: November 2006

A case of primary cutaneous zygomycosis caused by
Mucor circinelloides
is described. Histopathology showed typical hyphae along with chlamydospores. The isolate was identified by molecular and phenotypic methods. The utility of sequence analysis of the internal transcribed spacer region is highlighted; however, further studies are needed to assess species genetic heterogeneity.