bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering of Artificial Plant Cytochrome P450 Enzymes for Synthesis of Isoflavones by
Escherichia coli
Authors: Leonard Effendi , Koffas Mattheos A. G.
Published: September 2007

Engineered microbes are becoming increasingly important as recombinant production platforms. However, the nonfunctionality of membrane-bound cytochrome P450 enzymes precludes the use of industrially relevant prokaryotes such as
Escherichia coli
for high-level in vivo synthesis of many functional plant-derived compounds. We describe the design of a series of artificial isoflavone synthases that allowed the robust production of plant estrogen pharmaceuticals by
E. coli
. Through this methodology, a plant P450 construct was assembled to mimic the architecture of a self-sufficient bacterial P450 and contained tailor-made membrane recognition signals. The specific in vivo production catalyzed by one identified chimera was up to 20-fold higher than that achieved by the native enzyme expressed in a eukaryotic host and up to 10-fold higher than production by plants. This novel biological device is a strategy for the utilization of laboratory bacteria to robustly manufacture high-value plant P450 products.
Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from
Sphingomonas
sp. Strain CHY-1
Authors: Jouanneau Yves , Micoud Julien , Meyer Christine
Published: September 2007

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from
Sphingomonas
sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an α
3
β
3
heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent

K
m

for salicylate of 1.1 ± 0.2 μM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (

K
m

, 28 ± 6 μM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a

K
i

of 0.68 μM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.
Detection of
Histoplasma
Antigen by a Quantitative Enzyme Immunoassay
Authors: Connolly Patricia A. , Durkin Michelle M. , LeMonte Ann M. , Hackett Emily J. , Wheat L. Joseph
Published: October 2007

The second-generation
Histoplasma
antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of
Histoplasma
antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative
Histoplasma
antigen immunoassay.
High Prevalence of the CD14-159CC Genotype in Patients Infected with Severe Acute Respiratory Syndrome-Associated Coronavirus
Authors: Yuan Fang F. , Boehm Ingrid , Chan Paul K. S. , Marks Katherine , Tang Julian W. , Hui David S. C. , Sung Joseph J. Y. , Dyer Wayne B. , Geczy Andrew F. , Sullivan John S.
Published: October 2007

To investigate whether genetic factors of innate immunity might influence susceptibility and/or progression in individuals infected with SARS, we evaluated the CD14 gene polymorphism in 198 Hong Kong blood donors and 152 Hong Kong severe acute respiratory syndrome (SARS) patients who were previously genotyped for FcγRIIA polymorphisms. The prevalence of the CD14-159CC polymorphism was significantly higher in the patients with severe SARS than in the those with mild SARS or controls (31% versus 15% [mild SARS] or 20% [controls]; mild SARS:
P
= 0.029; odds ratio, 2.74; 95% confidence interval, 1.15 to 6.57; controls,
P
= 0.04; odds ratio, 2.41; 95% confidence interval, 1.05 to 5.54), and both CD14-159CC and FcγRIIA-RR131 are risk genotypes for severe SARS-CoV infection.
Immunity to
Neisseria meningitidis
Group B in Adults despite Lack of Serum Bactericidal Antibody
Authors: Welsch Jo Anne , Granoff Dan
Published: October 2007

Serum-complement-mediated bactericidal antibody (SBA) remains the serologic hallmark of protection against meningococcal disease, despite experimental and epidemiologic data that SBA may underestimate immunity. We measured bactericidal activity against three strains of
Neisseria meningitidis
group B in sera from 48 healthy adults and in whole blood from 15 subjects. Blood was anticoagulated with lepirudin, a specific thrombin inhibitor not known to activate complement. Depending on the test strain, protective SBA titers of ≥1:4 were present in only 8 to 15% of the subjects, whereas bactericidal activity was present in 40 to 87% of subjects according to the blood assay. Among SBA-negative subjects, blood from 23 to 42% gave a decrease of ≥2 log
10
CFU/ml after 1 h of incubation, and blood from 36 to 83% gave a decrease of ≥1 log
10
after 2 h. For most blood samples, bactericidal antibodies primarily were directed against noncapsular antigens, since activity was not inhibited by group B polysaccharide. For some SBA-negative subjects, white cells were not needed, since similar respective bactericidal activities were observed in blood and plasma. Bactericidal activity by whole blood of SBA-negative subjects can be rapid (<1 h) and effective (≥2 log
10
) and, among all subjects, was four- to sixfold more prevalent than a positive SBA. Thus, while an SBA titer of ≥1:4 predicts protection against meningococcal disease, a titer of <1:4 is poorly predictive of susceptibility. More sensitive assays than SBA are needed to assess protective meningococcal immunity, or we risk underestimating the extent of immunity in the population and the effectiveness of new meningococcal vaccines.
Developmental Commitment in
Dictyostelium discoideum
Authors: Katoh Mariko , Chen Guokai , Roberge Emily , Shaulsky Gad , Kuspa Adam
Published: September 2007

Upon starvation,
Dictyostelium discoideum
cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing
Dictyostelium
cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells’ inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells’ loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing
Dictyostelium
cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.
Alternative Modes of Organellar Segregation during Sporulation in
Saccharomyces cerevisiae
Authors: Suda Yasuyuki , Nakanishi Hideki , Mathieson Erin M. , Neiman Aaron M.
Published: September 2007

Formation of ascospores in the yeast
Saccharomyces cerevisiae
is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.
Candida albicans
Sun41p, a Putative Glycosidase, Is Involved in Morphogenesis, Cell Wall Biogenesis, and Biofilm Formation
Authors: Hiller Ekkehard , Heine Sonja , Brunner Herwig , Rupp Steffen
Published: September 2007

The SUN gene family has been defined in
Saccharomyces cerevisiae
and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In
Candida albicans
the SUN family comprises two genes,
SUN41
and
SIM1
. We demonstrate that
C. albicans
mutants lacking
SUN41
show similar defects as found for
S. cerevisiae
, including defects in cytokinesis. In addition, the
SUN41
mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type,
SUN41
deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.
Genome-Wide Expression and Location Analyses of the
Candida albicans
Tac1p Regulon
Authors: Liu Teresa T. , Znaidi Sadri , Barker Katherine S. , Xu Lijing , Homayouni Ramin , Saidane Saloua , Morschhäuser Joachim , Nantel André , Raymond Martine , Rogers P. David
Published: September 2007

A major mechanism of azole resistance in
Candida albicans
is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of
CDR1
- and
CDR2
-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with
CDR1
and
CDR2
, including
TAC1
itself, and 12 consistently down-regulated genes. When a resistant strain deleted for
TAC1
was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including
CDR1
and
CDR2
. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN
4
CGG), including
TAC1
. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a
TAC1
-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets:
CDR1, CDR2, GPX1
(putative glutathione peroxidase),
LCB4
(putative sphingosine kinase),
RTA3
(putative phospholipid flippase), and orf19.1887 (putative lipase), as well as
IFU5
and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in
C. albicans
.
Reassessment of the In Vivo Functions of DNA Polymerase I and RNase H in Bacterial Cell Growth
Authors: Fukushima Sanae , Itaya Mitsuhiro , Kato Hiroaki , Ogasawara Naotake , Yoshikawa Hirofumi
Published: September 2007

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by
rnh
genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the
polA
and
rnh
genes. In a previous study, we looked for the synthetic lethality of paralogs in
Bacillus subtilis
and detected several essential doublet paralogs, including the
polA ypcP
pair. YpcP consists of only the 5′-3′ exonuclease domain. In the current study, we first confirmed that the
polA
genes of both
Escherichia coli
and
B. subtilis
could be completely deleted. We found that the 5′-3′ exonuclease activity encoded by either
polA
or
ypcP xni
was required for the growth of
B. subtilis
and
E. coli
. Also, the 5′-3′ exonuclease activity of Pol I was indispensable in the cyanobacterium
Synechococcus elongatus
. These results suggest that a 5′-3′ exonuclease activity is essential in these organisms. Our success in constructing a
B. subtilis
strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5′-3′ exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5′-3′ exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.
Characterization of an Active Partition System for theEnterococcus faecalisPheromone-Responding Plasmid pAD1
Authors: Francia Maria Victoria , Weaver Keith E. , Goicoechea Patricia , Tille Patricia , Clewell Don B.
Published: September 2007

Enterococcus faecalisplasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol.175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol.186:5003-5016, 2004). The present study focuses on the adjacent determinantsrepBandrepC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribedrepAandrepB. Through mutagenesis andtrans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active intrans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of therepBClocus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.
Atomic Force Microscopy Study of the Effect of Lipopolysaccharides and Extracellular Polymers on Adhesion of
Pseudomonas aeruginosa
Authors: Atabek Arzu , Camesano Terri A.
Published: September 2007

The roles of lipopolysaccharides (LPS) and extracellular polymers (ECP) on the adhesion of
Pseudomonas aeruginosa
PAO1 (expresses the A-band and B-band of O antigen) and AK1401 (expresses the A-band but not the B-band) to silicon were investigated with atomic force microscopy (AFM) and related to biopolymer physical properties. Measurement of macroscopic properties showed that strain AK1401 is more negatively charged and slightly more hydrophobic than strain PAO1 is. Microscopic AFM investigations of individual bacteria showed differences in how the biopolymers interacted with silicon. PAO1 showed larger decay lengths in AFM approach cycles, suggesting that the longer polymers on PAO1 caused greater steric repulsion with the AFM tip. For both bacterial strains, the long-range interactions we observed (hundreds of nanometers) were inconsistent with the small sizes of LPS, suggesting that they were also influenced by ECP, especially polysaccharides. The AFM retraction profiles provide information on the adhesion strength of the biopolymers to silicon (
F
adh
). For AK1401, the adhesion forces were only slightly lower (
F
adh
= 0.51 nN compared to 0.56 nN for PAO1), but the adhesion events were concentrated over shorter distances. More than 90% of adhesion events for AK1401 were at distances of <600 nm, while >50% of adhesion events for PAO1 were at distances of >600 nm. The sizes of the observed molecules suggest that the adhesion of
P. aeruginosa
to silicon was controlled by ECP, in addition to LPS. Steric and electrostatic forces each contributed to the interfacial interactions between
P. aeruginosa
and the silicon surface.
Anaplasma phagocytophilum p44
mRNA Expression Is Differentially Regulated in Mammalian and Tick Host Cells: Involvement of the DNA Binding Protein ApxR
Authors: Wang Xueqi , Cheng Zhihui , Zhang Chunbin , Kikuchi Takane , Rikihisa Yasuko
Published: September 2007

The natural life cycle of
Anaplasma phagocytophilum
, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of
A. phagocytophilum p44
genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of
p44
mRNA obtained from spleens of
A. phagocytophilum
-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of
A. phagocytophilum
-infected
Ixodes scapularis
nymphs. Similarly, the amount of
p44
mRNA obtained from
A. phagocytophilum
-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of
p44
mRNA was approximately threefold higher in
A. phagocytophilum
-infected HL-60 cells cultured at 37°C than in
A. phagocytophilum
-infected HL-60 cells cultured at 28°C. Although there are more than 100
p44
paralogs, we observed expression mainly from the
p44
expression locus (
p44E
) in various host environments. Interestingly, transcription of the
A. phagocytophilum
gene encoding the DNA binding protein ApxR was also significantly greater in
A. phagocytophilum
-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of
p44E
and
apxR
. ApxR also transactivated the
p44E
and
apxR
promoter regions in a
lacZ
reporter assay. These results indicate that
p44
genes and
apxR
are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of
p44E
.
TheMyxococcus xanthusNla4 Protein Is Important for Expression of Stringent Response-Associated Genes, ppGpp Accumulation, and Fruiting Body Development
Authors: Ossa Faisury , Diodati Michelle E. , Caberoy Nora B. , Giglio Krista M. , Edmonds Mick , Singer Mitchell , Garza Anthony G.
Published: September 2007

Changes in gene expression are important for the landmark morphological events that occur duringMyxococcus xanthusfruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP genenla4affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that thenla4mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that thenla4mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed thatnla4cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including theM. xanthusppGpp synthetase generelA, are altered innla4mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.
Polymerization of the Actin-Like Protein MamK, Which Is Associated with Magnetosomes
Authors: Taoka Azuma , Asada Ryuji , Wu Long-Fei , Fukumori Yoshihiro
Published: September 2007

The recombinant actin-like protein MamK was purified from
Escherichia coli
and used as an antigen to generate the anti-MamK antibody. Immunostaining studies showed a linear distribution of MamK in
Magnetospirillum magnetotacticum
cells and of MamK in association with magnetosomes. Moreover, we demonstrated that MamK polymerizes into filamentous bundles in vitro.
The Wetting Agent Required for Swarming in
Salmonella enterica
Serovar Typhimurium Is Not a Surfactant
Authors: Chen Bryan G. , Turner Linda , Berg Howard C.
Published: September 2007

We compared the abilities of media from agar plates surrounding swarming and nonswarming cells of
Salmonella enterica
serovar Typhimurium to wet a nonpolar surface by measuring the contact angles of small drops. The swarming cells were wild type for chemotaxis, and the nonswarming cells were nonchemotactic mutants with motor biases that were counterclockwise (
cheY
) or clockwise (
cheZ
). The latter strains have been shown to be defective for swarming because the agar remains dry (Q. Wang, A. Suzuki, S. Mariconda, S. Porwollik, and R. M. Harshey, EMBO J. 24:2034-2042, 2005). We found no differences in the abilities of the media surrounding these cells, either wild type or mutant, to wet a low-energy surface (freshly prepared polydimethylsiloxane); although, their contact angles were smaller than that of the medium harvested from the underlying agar. So the agent that promotes wetness produced by wild-type cells is not a surfactant; it is an osmotic agent.
Population Genetic Structure of the
Staphylococcus intermedius
Group: Insights into
agr
Diversification and the Emergence of Methicillin-Resistant Strains
Authors: Bannoehr Jeanette , Ben Zakour Nouri L. , Waller Andrew S. , Guardabassi Luca , Thoday Keith L. , van den Broek Adri H. M. , Fitzgerald J. Ross
Published: September 2007

The population genetic structure of the animal pathogen
Staphylococcus intermedius
is poorly understood. We carried out a multilocus sequence phylogenetic analysis of isolates from broad host and geographic origins to investigate inter- and intraspecies diversity. We found that isolates phenotypically identified as
S. intermedius
are differentiated into three closely related species,
S. intermedius
,
Staphylococcus pseudintermedius
, and
Staphylococcus delphini. S. pseudintermedius
, not
S. intermedius
, is the common cause of canine pyoderma and occasionally causes zoonotic infections of humans. Over 60 extant STs were identified among the
S. pseudintermedius
isolates examined, including several that were distributed on different continents. As the
agr
quorum-sensing system of staphylococci is thought to have evolved along lines of speciation within the genus, we examined the allelic variation of
agrD
, which encodes the autoinducing peptide (AIP). Four AIP variants were encoded by
S. pseudintermedius
isolates, and identical AIP variants were shared among the three species, suggesting that a common quorum-sensing capacity has been conserved in spite of species differentiation in largely distinct ecological niches. A lack of clonal association of
agr
alleles suggests that assortive recombination may have contributed to the distribution of
agr
diversity. Finally, we discovered that the recent emergence of methicillin-resistant strains was due to multiple acquisitions of the
mecA
gene by different
S. pseudintermedius
clones found on different continents. Taken together, these data have resolved the population genetic structure of the
S. intermedius
group, resulting in new insights into its ancient and recent evolution.
DNA Bending in the Mycobacterial Plasmid pAL5000 Origin-RepB Complex
Authors: Chatterjee Sujoy , Basu Arnab , Basu Abhijit , Das Gupta Sujoy K.
Published: September 2007

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in gram-positiveActinomycetesbacteria. The replication regions of these plasmids comprise a bicistronic operon,repA-repB, encoding two replication proteins. Located upstream is acis-acting element that functions as the origin of replication. It comprises an ∼200-bp segment spanning two binding sites for the replication protein RepB, a low-affinity (L) site and a high-affinity (H) site separated by an ∼40-bp spacer sequence. The trajectory of the DNA in the RepB-origin complex has been investigated, and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long-range polar curvature which affected the DNA sequences 3′ to the H site. These movements appear to be essential for the in-phase alignment of the L and H sites that leads to the formation of a looped structure. A novel property of RepB unearthed in this study is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origins of replication of pAL5000 and related plasmids are highly flexible and that multimeric, RepB-like initiator proteins bind the origin and induce local deformations and long-range curvatures which are probably necessary for the proper functioning of the origin.
The GATC-Binding Protein SeqA Is Required for Bile Resistance and Virulence inSalmonella entericaSerovar Typhimurium
Authors: Prieto Ana I. , Jakomin Marcello , Segura Ignacio , Pucciarelli M. Graciela , Ramos-Morales Francisco , García-del Portillo Francisco , Casadesús Josep
Published: September 2007

Disruption of theseqAgene ofSalmonella entericaserovar Typhimurium causes defects similar to those described inE. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA−mutants ofE. coliandS. enterica, however, are found. SeqA−mutants ofS. entericaform normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation ofS. entericavirulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA−mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA−mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.
Phenotypic Characterization of Pore Mutants of theVibrio choleraePorin OmpU
Authors: Pagel Melissa , Simonet Valérie , Li Jie , Lallemand Mathilde , Lauman Brian , Delcour Anne H.
Published: September 2007

General-diffusion porins form large β-barrel channels that control the permeability of the outer membrane of gram-negative bacteria to nutrients, some antibiotics, and external signals. Here, we have analyzed the effects of mutations in the OmpU porin ofVibrio choleraeat conserved residues that are known to affect pore properties in theEscherichia coliporins OmpF and OmpC. Various phenotypes were investigated, including sensitivity to β-lactam antibiotics, growth on large sugars, and sensitivity to and biofilm induction by sodium deoxycholate, a major bile component that acts as an external signal for multiple cellular responses of this intestinal pathogen. Overall, our results indicate that specific residues play different roles in controlling the passage of various compounds. Mutations of barrel wall arginine residues that protrude in the pore affect pore size and growth in the presence of large sugars or sodium deoxycholate. Sensitivity to large cephalosporins is mostly affected by D116, located on the L3 loop, whose homolog inE. coli, OmpF, is a known binding determinant for these drugs. L3 loop residues also affect biofilm induction. The results are interpreted in terms of a homology model based on the structures ofE. coliporins.
The RcsCDB Signaling System and Swarming Motility inSalmonella entericaSerovar Typhimurium: Dual Regulation of Flagellar and SPI-2 Virulence Genes
Authors: Wang Qingfeng , Zhao Yifang , McClelland Michael , Harshey Rasika M.
Published: September 2007

The Rcs phosphorelay is a multicomponent signaling system that positively regulates colanic acid synthesis and negatively regulates motility and virulence. We have exploited a spontaneously isolated mutant, IgaA(T191P), that is nearly maximally activated for the Rcs system to identify a vast set of genes that respond to the stimulation, and we report new regulatory properties of this signaling system inSalmonella entericaserovar Typhimurium. Microarray data show that the Rcs system normally functions as a positive regulator of SPI-2 and other genes important for the growth ofSalmonellain macrophages, although when highly activated the system completely represses the SPI-1/SPI-2 virulence, flagellar, and fimbrial biogenesis pathways. The auxiliary protein RcsA, which works with RcsB to positively regulate colanic acid and other target genes, not only stimulates but also antagonizes the positive regulation of many genes in theigaAmutant. We show that RcsB represses motility through the RcsB box in the promoter region of the master operonflhDCand that RcsA is not required for this regulation. Curiously, RcsB selectively stimulates expression of the flagellar type 3 secretion genesfliPQR; an RcsAB box located downstream offliRinfluences this regulation. We show that excess colanic acid impairs swimming and inhibits swarming motility, consistent with the inverse regulation of the two pathways by the Rcs system.
Dynamics ofEscherichia coliChromosome Segregation during Multifork Replication
Authors: Nielsen Henrik J. , Youngren Brenda , Hansen Flemming G. , Austin Stuart
Published: September 2007

Slowly growingEscherichia colicells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.
Genetic Dissection of the Divergent Activities of the Multifunctional Membrane Sensor BglF
Authors: Monderer-Rothkoff Galya , Amster-Choder Orna
Published: September 2007

BglF catalyzes β-glucoside phosphotransfer across the cytoplasmic membrane inEscherichia coli. In addition, BglF acts as a sugar sensor that controls expression of β-glucoside utilization genes by reversibly phosphorylating the transcriptional antiterminator BglG. Thus, BglF can exist in two opposed states: a nonstimulated state that inactivates BglG by phosphorylation and a sugar-stimulated state that activates BglG by dephosphorylation and phosphorylates the incoming sugar. Sugar phosphorylation and BglG (de)phosphorylation are both catalyzed by the same residue, Cys24. To investigate the coordination and the structural requirements of the opposing activities of BglF, we conducted a genetic screen that led to the isolation of mutations that shift the balance toward BglG phosphorylation. We show that some of the mutants that are impaired in dephosphorylation of BglG retained the ability to catalyze the concurrent activity of sugar phosphotransfer. These mutations map to two regions in the BglF membrane domain that, based on their predicted topology, were suggested to be implicated in activity. Using in vivo cross-linking, we show that a glycine in the membrane domain, whose substitution impaired the ability of BglF to dephosphorylate BglG, is spatially close to the active-site cysteine located in a hydrophilic domain. This residue is part of a newly identified motif conserved among β-glucoside permeases associated with RNA-binding transcriptional antiterminators. The phenotype of the BglF mutants could be suppressed by BglG mutants that were isolated by a second genetic screen. In summary, we identified distinct sites in BglF that are involved in regulating phosphate flow via the common active-site residue in response to environmental cues.
Distribution of Centromere-LikeparSSites in Bacteria: Insights from Comparative Genomics
Authors: Livny Jonathan , Yamaichi Yoshiharu , Waldor Matthew K.
Published: September 2007

Partitioning of low-copy-number plasmids to daughter cells often depends on ParA and ParB proteins acting on centromere-likeparSsites. Similar chromosome-encodedparloci likely also contribute to chromosome segregation. Here, we used bioinformatic approaches to search for chromosomalparSsites in 400 prokaryotic genomes. Although the consensus sequence matrix used to search forparSsites was derived from two gram-positive species, putativeparSsites were identified on the chromosomes of 69% of strains from all branches of bacteria. Strains that were not found to containparSsites clustered among relatively few branches of the prokaryotic evolutionary tree. In the vast majority of cases,parSsites were identified in origin-proximal regions of chromosomes. The widespread conservation ofparSsites across diverse bacteria suggests thatparloci evolved very early in the evolution of bacterial chromosomes and that the absence ofparS,parA, and/orparBin certain strains likely reflects the loss of one of more of these loci much later in evolution. Moreover, the highly conserved origin-proximal position ofparSsuggestsparloci are primarily devoted to regulating processes that involve the origin region of bacterial chromosomes. In species containing multiple chromosomes, theparSsites found on secondary chromosomes diverge significantly from those found on their primary chromosomes, suggesting that chromosome segregation of multipartite genomes requires distinct replicon-specificparloci. Furthermore,parSsites on secondary chromosomes are not well conserved among different species, suggesting that the evolutionary histories of secondary chromosomes are more diverse than those of primary chromosomes.
The
Myxococcus xanthus
Developmental Program Can Be Delayed by Inhibition of DNA Replication
Authors: Rosario Christopher J. , Singer Mitchell
Published: September 2007

Under conditions of nutrient deprivation,
Myxococcus xanthus
undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive
dnaB
mutant, DnaB
A116V
, was isolated from
M. xanthus
. Unlike what happens in
Escherichia coli dnaB
mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the
M. xanthus
mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB
A116V
mutant arrested as a population of 1
n
cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB
A116V
mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.
Molecular Typing of a Suspected Cluster of
Nocardia farcinica
Infections by Use of Randomly Amplified Polymorphic DNA, Pulsed-Field Gel Electrophoresis, and Amplified Fragment Length Polymorphism Analyses
Authors: Kalpoe J. S. , Templeton K. E. , Horrevorts A. M. , Endtz H. P. , Kuijper E. J. , Bernards A. T. , Klaassen C. H. W.
Published: October 2007

Randomly amplified polymorphic DNA (RAPD), pulsed-field gelelectrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analyses were used to investigate a possible outbreak of
Nocardia farcinica
. RAPD and PFGE analyses yielded irreproducible and unsatisfactory results, respectively. AFLP analysis seem to be a promising and welcome addition for molecular analysis of
Nocardia
isolates.
Mycobacterium avium
Subspecies
paratuberculosis
Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities
Authors: Scanu Antonio M. , Bull Tim J. , Cannas Sara , Sanderson Jeremy D. , Sechi Leonardo A. , Dettori Giuseppe , Zanetti Stefania , Hermon-Taylor John
Published: October 2007

Mycobacterium avium
subsp.
paratuberculosis
causes Johne's disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johne's disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between
Mycobacterium avium
subsp.
paratuberculosis
infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohn's disease and healthy control groups were also included.
Mycobacterium avium
subsp.
paratuberculosis
was detected by IS
900
PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained.
Mycobacterium avium
subsp.
paratuberculosis
was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohn's disease (
P
= 0.0003 for irritable bowel syndrome patients versus healthy controls and
P
= 0.0000 for Crohn's disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS
900
that was also found in isolates from seven of eight dairy sheep. There was a significant association (
P
= 0.0018) between
Mycobacterium avium
subsp.
paratuberculosis
infection and the consumption of hand-made cheese.
Mycobacterium avium
subsp.
paratuberculosis
is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohn's disease.
Emergence of Usutu Virus in Hungary
Authors: Bakonyi Tamás , Erdélyi Károly , Ursu Krisztina , Ferenczi Emőke , Csörgő Tibor , Lussy Helga , Chvala Sonja , Bukovsky Christiane , Meister Tanja , Weissenböck Herbert , Nowotny Norbert
Published: October 2007

In 2001, Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus serogroup related to West Nile virus and previously restricted to sub-Saharan Africa, emerged in wild and zoo birds in and around Vienna, Austria. In order to monitor the spread of the infection, a dead bird surveillance program was established in Austria and in neighboring Hungary. In Hungary, 332 dead birds belonging to 52 species were tested for USUV infection between 2003 and 2006. In the first 2 years, all birds investigated were negative. In August 2005, however, USUV was detected in organ samples of a blackbird (
Turdus merula
), which was found dead in Budapest, Hungary, by reverse transcription-PCR, immunohistochemistry, and in situ hybridization. In July and August 2006, a further six dead blackbirds tested positive for USUV, and the virus was isolated from organ samples of one bird. These birds were also found in urban areas of Budapest. The nearly complete genomic sequence of one Hungarian USUV strain was determined; it was found to share 99.9% identity with the strain that has been circulating in Austria since 2001. This result indicates that the USUV strain responsible for the blackbird die-off in Budapest most likely spread from Austria to Hungary instead of being independently introduced from Africa.
Human Seminal Plasma Abrogates the Capture and Transmission of Human Immunodeficiency Virus Type 1 to CD4
+
T Cells Mediated by DC-SIGN
Authors: Sabatté Juan , Ceballos Ana , Raiden Silvina , Vermeulen Mónica , Nahmod Karen , Maggini Julián , Salamone Gabriela , Salomón Horacio , Amigorena Sebastian , Geffner Jorge
Published: October 2007

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is expressed by dendritic cells (DCs) at mucosal surfaces and appears to play an important role in the dissemination of human immunodeficiency virus type 1 (HIV-1) infection. DC-SIGN binds HIV-1 gp120 and efficiently transmits the virus to T CD4
+
cells, which become the center of viral replication. Semen represents the main vector for HIV-1 dissemination worldwide. In the present study we show that human seminal plasma (SP), even when used at very high dilutions (1:10
4
to 1:10
5
), markedly inhibits the capture and transmission of HIV-1 to T CD4
+
cells mediated by both DCs and B-THP-1-DC-SIGN cells. In contrast, SP does not inhibit the capture of HIV-1 by DC-SIGN-negative target cells, such as the T-cell line SupT-1, monocytes, and activated peripheral blood mononuclear cells. The SP inhibitor has a high molecular mass (>100 kDa) and directly interacts with DC-SIGN-positive target cells but not with HIV-1. Moreover, the inhibitor binds to concanavalin A, suggesting that it contains high-mannose N-linked carbohydrates. Of note, using biotin-labeled SP we found that the binding of SP components to DCs was abrogated by mannan, while their interaction with B-THP-1 cells was almost completely dependent on the expression of DC-SIGN. Since epithelium integrity is often compromised after vaginal or anal intercourse, as well as in the presence of ulcerative-sexually transmitted diseases, our results support the notion that components of the SP might be able to access to the subepithelium, inhibiting the recognition of HIV-1 gp120 by DC-SIGN-positive DCs.