bluuebunny/crossref_metadata_2025 huggingface dataset:
Biodegradation of Bis(2-Chloroethyl) Ether by
Xanthobacter
sp. Strain ENV481
Authors: McClay Kevin , Schaefer Charles E. , Vainberg Simon , Steffan Robert J.
Published: September 2007

Degradation of bis(2-chloroethyl) ether (BCEE) was observed to occur in two bacterial strains. Strain ENV481, a
Xanthobacter
sp. strain, was isolated by enrichment culturing of samples from a Superfund site located in the northeastern United States. The strain was able to grow on BCEE or 2-chloroethylethyl ether as the sole source of carbon and energy. BCEE degradation in strain ENV481 was facilitated by sequential dehalogenation reactions resulting in the formation of 2-(2-chloroethoxy)ethanol and diethylene glycol (DEG), respectively. 2-Hydroxyethoxyacetic acid was detected as a product of DEG catabolism by the strain. Degradation of BCEE by strain ENV481 was independent of oxygen, and the strain was not able to grow on a mixture of benzene, ethylbenzene, toluene, and xylenes, other prevalent contaminants at the site. Another bacterial isolate,
Pseudonocardia
sp. strain ENV478 (S. Vainberg et al., Appl. Environ. Microbiol. 72:5218-5224, 2006), degraded BCEE after growth on tetrahydrofuran or propane but was not able to grow on BCEE as a sole carbon source. BCEE degradation by strain ENV478 appeared to be facilitated by a monooxygenase-mediated O-dealkylation mechanism, and it resulted in the accumulation of 2-chloroacetic acid that was not readily degraded by the strain.
Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications
Authors: Löfblom John , Sandberg Julia , Wernérus Henrik , Ståhl Stefan
Published: September 2007

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (

K
D

) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both

K
D

and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.
Effects of Plant Biomass, Plant Diversity, and Water Content on Bacterial Communities in Soil Lysimeters: Implications for the Determinants of Bacterial Diversity
Authors: Zul Delita , Denzel Sabine , Kotz Andrea , Overmann Jörg
Published: September 2007

Soils may comprise tens of thousands to millions of bacterial species. It is still unclear whether this high level of diversity is governed by functional redundancy or by a multitude of ecological niches. In order to address this question, we analyzed the reproducibility of bacterial community composition after different experimental manipulations. Soil lysimeters were planted with four different types of plant communities, and the water content was adjusted. Group-specific phylogenetic fingerprinting by PCR-denaturing gradient gel electrophoresis revealed clear differences in the composition of
Alphaproteobacteria
,
Betaproteobacteria
,
Bacteroidetes
,
Chloroflexi
,
Planctomycetes
, and
Verrucomicrobia
populations in soils without plants compared to that of populations in planted soils, whereas no influence of plant species composition on bacterial diversity could be discerned. These results indicate that the presence of higher plant species affects the species composition of bacterial groups in a reproducible manner and even outside of the rhizosphere. In contrast, the environmental factors tested did not affect the composition of
Acidobacteria
,
Actinobacteria
,
Archaea
, and
Firmicutes
populations. One-third (52 out of 160) of the sequence types were found to be specifically and reproducibly associated with the absence or presence of plants. Unexpectedly, this was also true for numerous minor constituents of the soil bacterial assemblage. Subsequently, one of the low-abundance phylotypes (beta10) was selected for studying the interdependence under particular experimental conditions and the underlying causes in more detail. This so-far-uncultured phylotype of the
Betaproteobacteria
species represented up to 0.18% of all bacterial cells in planted lysimeters compared to 0.017% in unplanted systems. A cultured representative of this phylotype exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. Our results suggest that the bacterial species composition in soil is determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches.
An Inducible Propane Monooxygenase Is Responsible for
N
-Nitrosodimethylamine Degradation by
Rhodococcus
sp. Strain RHA1
Authors: Sharp Jonathan O. , Sales Christopher M. , LeBlanc Justin C. , Liu Jie , Wood Thomas K. , Eltis Lindsay D. , Mohn William W. , Alvarez-Cohen Lisa
Published: September 2007

Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant
N
-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that
Rhodococcus
sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of
prmA
, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane.
Genetic Method To Analyze Essential Genes of
Escherichia coli
Authors: Hupert-Kocurek Katarzyna , Sage Jay M. , Makowska-Grzyska Magdalena , Kaguni Jon M.
Published: September 2007

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the
dnaC
gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of
Escherichia coli
and related enterobacteria. The method consists of using a strain carrying a large deletion of the
dnaC
gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-
d
-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a
dnaC
mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.
The Fungus-Specific HET Domain Mediates Programmed Cell Death in
Podospora anserina
Authors: Paoletti M. , Clavé C.
Published: September 2007

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (
het
genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic
het-C
/
het-E
incompatibility system is the best-characterized incompatibility system in
Podospora anserina
. Cell death is triggered by interaction of specific alleles of
het-C
, encoding a glycolipid transfer protein, and
het-E
, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from
het-E
results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of
het-C
. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.
Expression of Hygromycin Phosphotransferase Alters Virulence of
Histoplasma capsulatum
Authors: Smulian A. George , Gibbons Reta S. , Demland Jeffery A. , Spaulding Deborah T. , Deepe George S.
Published: September 2007

The
Escherichia coli
hygromycin phosphotransferase (
hph
) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the
hph
gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of
Histoplasma capsulatum
in the generation of knockout strains of
H. capsulatum
or as a marker in mutant strains.
hph
-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the
hph
gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of
H. capsulatum
. Infection of mice with
hph
-expressing
H. capsulatum
yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the
hph
construct into the genomes or to the method of integration and was not
H. capsulatum
strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the
hph
gene.
Supramolecular Organization of the Respiratory Chain in
Neurospora crassa
Mitochondria
Authors: Marques Isabel , Dencher Norbert A. , Videira Arnaldo , Krause Frank
Published: September 2007

The existence of specific respiratory supercomplexes in mitochondria of most organisms has gained much momentum. However, its functional significance is still poorly understood. The availability of many deletion mutants in complex I (NADH:ubiquinone oxidoreductase) of
Neurospora crassa
, distinctly affected in the assembly process, offers unique opportunities to analyze the biogenesis of respiratory supercomplexes. Herein, we describe the role of complex I in assembly of respiratory complexes and supercomplexes as suggested by blue and colorless native polyacrylamide gel electrophoresis and mass spectrometry analyses of mildly solubilized mitochondria from the wild type and eight deletion mutants. As an important refinement of the fungal respirasome model, we found that the standard respiratory chain of
N. crassa
comprises putative complex I dimers in addition to I-III-IV and III-IV supercomplexes. Three
Neurospora
mutants able to assemble a complete complex I, lacking only the disrupted subunit, have respiratory supercomplexes, in particular I-III-IV supercomplexes and complex I dimers, like the wild-type strain. Furthermore, we were able to detect the I-III-IV supercomplexes in the nuo51 mutant with no overall enzymatic activity, representing the first example of inactive respirasomes. In addition, III-IV supercomplexes were also present in strains lacking an assembled complex I, namely, in four membrane arm subunit mutants as well as in the peripheral arm nuo30.4 mutant. In membrane arm mutants, high-molecular-mass species of the 30.4-kDa peripheral arm subunit comigrating with III-IV supercomplexes and/or the prohibitin complex were detected. The data presented herein suggest that the biogenesis of complex I is linked with its assembly into supercomplexes.
Novel Membrane-Bound eIF2α Kinase in the Flagellar Pocket of
Trypanosoma brucei
Authors: Moraes Maria Carolina S. , Jesus Teresa C. L. , Hashimoto Nilce N. , Dey Madhusudan , Schwartz Kevin J. , Alves Viviane S. , Avila Carla C. , Bangs James D. , Dever Thomas E. , Schenkman Sergio , Castilho Beatriz A.
Published: September 2007

Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations.
Trypanosoma brucei
, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes.
T. brucei
eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in
T. brucei
, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes.
A Chemogenomic Screening of Sulfanilamide-Hypersensitive
Saccharomyces cerevisiae
Mutants Uncovers
ABZ2
, the Gene Encoding a Fungal Aminodeoxychorismate Lyase
Authors: Botet Javier , Mateos Laura , Revuelta José L. , Santos María A.
Published: September 2007

Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the
p
-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (
YHI9
and
YMR289w
) were also identified. A detailed characterization of
YMR289w
revealed that this gene was required for growth in media lacking
p
-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for
p
-aminobenzoic acid biosynthesis. In light of these results,
YMR289w
was designated
ABZ2
, in accordance with the accepted nomenclature.
ABZ2
was able to rescue the
p
-aminobenzoate auxotrophy of an
Escherichia coli pabC
mutant, thus demonstrating that
ABZ2
and
pabC
are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.
Small Trypanosome RNA-Binding Proteins
Tb
UBP1 and
Tb
UBP2 Influence Expression of F-Box Protein mRNAs in Bloodstream Trypanosomes
Authors: Hartmann Claudia , Benz Corinna , Brems Stefanie , Ellis Louise , Luu Van-Duc , Stewart Mhairi , D'Orso Iván , Busold Christian , Fellenberg Kurt , Frasch Alberto C. C. , Carrington Mark , Hoheisel Jörg , Clayton Christine E.
Published: September 2007

In the African trypanosome
Trypanosoma brucei
nearly all control of gene expression is posttranscriptional; sequences in the 3′-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins,
Tb
UBP1 and
Tb
UBP2, containing a single RNA recognition motif, in trypanosome gene expression.
Tb
UBP1 and
Tb
UBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of
Tb
UBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the
CFB1
mRNA, which encodes a protein with a cyclin F-box domain.
CFB1
regulation was mediated by the 3′-untranslated region and involved stabilization of the mRNA. Depletion of
Tb
UBP2 and
Tb
UBP1 inhibited growth and downregulated expression of the cyclin F box protein gene
CFB2
;
trans
splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to
Tb
UBP2 or
Tb
UBP1, with some preference for
CFB1
. We suggest that
Tb
UBP1 and
Tb
UBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.
Requirement for
Candida albicans
Sun41 in Biofilm Formation and Virulence
Authors: Norice Carmelle T. , Smith Frank J. , Solis Norma , Filler Scott G. , Mitchell Aaron P.
Published: September 2007

The cell wall of
Candida albicans
lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in
SUN41
, Orf19.5412, Orf19.1277,
MSB2
, Orf19.3869, and
WSC1
caused hypersensitivity to the cell wall inhibitor caspofungin, while two different
ecm33
insertions caused mild caspofungin resistance. Insertion mutations in
SUN41
and Orf19.5412 caused biofilm defects. Through analysis of homozygous
sun41
Δ/
sun41
Δ deletion mutants and
sun41
Δ/
sun41
Δ+p
SUN41
-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The
sun41
Δ/
sun41
Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the
sun41
Δ/
sun41
Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.
Control of Cell Wall Assembly by a Histone-Like Protein in Mycobacteria
Authors: Katsube Tomoya , Matsumoto Sohkichi , Takatsuka Masaki , Okuyama Megumi , Ozeki Yuriko , Naito Mariko , Nishiuchi Yukiko , Fujiwara Nagatoshi , Yoshimura Mamiko , Tsuboi Takafumi , Torii Motomi , Oshitani Nobuhide , Arakawa Tetsuo , Kobayashi Kazuo
Published: September 2007

Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient
Mycobacterium smegmatis
strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.
Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted
Enterococcus faecium
Clonal Complex 17 Isolates
Authors: Hendrickx Antoni P. A. , van Wamel Willem J. B. , Posthuma George , Bonten Marc J. M. , Willems Rob J. L.
Published: September 2007

Most
Enterococcus faecium
isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17
E. faecium
. Using PCR and Southern and dot blot hybridizations, 131
E. faecium
isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the
E. faecium
TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in
E. faecium
CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (
P
< 0.05). Three of these CWAP genes clustered together on the
E. faecium
TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering
E. faecium
contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.
Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Tzianabos Arthur O. , Smith C. Jeffrey
Published: September 2007

Results of this study showed that the anaerobic, opportunistic pathogen
Bacteroides fragilis
lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the
B. fragilis trxB
gene was induced following treatment with diamide or H
2
O
2
or exposure to oxygen. This inducible
trxB
expression was OxyR independent. Northern blot hybridization analysis showed that the
trxB
mRNA was cotranscribed with
lolA
as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A
trxB
deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the
trxB
mutant was unable to grow in culture media without addition of a reductant. Furthermore, the
trxB
mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.
Phosphopantetheine Adenylyltransferase from
Escherichia coli
: Investigation of the Kinetic Mechanism and Role in Regulation of Coenzyme A Biosynthesis
Authors: Miller J. Richard , Ohren Jeffrey , Sarver Ronald W. , Mueller W. Thomas , Dreu Piet de , Case Heather , Thanabal Venkataraman
Published: September 2007

Phosphopantetheine adenylyltransferase (PPAT) from
Escherichia coli
is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in
E. coli
, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.
Structure and Biological Activities of Beta Toxin fromStaphylococcus aureus
Authors: Huseby Medora , Shi Ke , Brown C. Kent , Digre Jeff , Mengistu Fikre , Seo Keun Seok , Bohach Gregory A. , Schlievert Patrick M. , Ohlendorf Douglas H. , Earhart Cathleen A.
Published: September 2007

Beta toxin is a neutral sphingomyelinase secreted by certain strains ofStaphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases ofListeria ivanoviiandBacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1,Escherichia coliendonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 fromBombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.
Sigma Factor Genes
sigC
,
sigE
, and
sigG
Are Upregulated in Heterocysts of the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Aldea M. Ramona , Mella-Herrera Rodrigo A. , Golden James W.
Published: September 2007

We used
gfp
transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium
Anabaena
sp. strain PCC 7120. Reporter strains containing
gfp
fusions with the upstream regions of
sigB2
,
sigD
,
sigI
, and
sigJ
did not show developmental regulation. Time-lapse microscopy of
sigC
,
sigE
, and
sigG
reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.
Distribution, Functional Expression, and Genetic Organization of Cif, a Phage-Encoded Type III-Secreted Effector from Enteropathogenic and EnterohemorrhagicEscherichia coli
Authors: Loukiadis Estelle , Nobe Rika , Herold Sylvia , Tramuta Clara , Ogura Yoshitoshi , Ooka Tadasuke , Morabito Stefano , Kérourédan Monique , Brugère Hubert , Schmidt Herbert , Hayashi Tetsuya , Oswald Eric
Published: September 2007

EnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049E. colistrains of human, animal, and environmental origins. A total of 115E. coliisolates from diverse origins and geographic locations carriedcif. The presence ofcifwas tightly associated with the LEE, since all thecif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population ofE. colibut positively selected within the population of LEE-positive strains. Nonetheless, 66% ofcif-positiveE. colistrains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in thecifgene. The passenger region of the prophages carryingcifwas highly variable and showed various combinations of IS elements and genes coding for other effectors such asnleB,nleC,nleH,nleG,espJ, andnleA/espI(some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.
A Functional Two-Partner Secretion System Contributes to Adhesion of
Neisseria meningitidis
to Epithelial Cells
Authors: Schmitt Corinna , Turner David , Boesl Maria , Abele Marion , Frosch Matthias , Kurzai Oliver
Published: September 2007

Neisseria meningitidis
is a frequent commensal of the human nasopharynx causing severe invasive infections in rare cases. A functional two-partner secretion (TPS) system in
N. meningitidis
, composed of the secreted effector protein HrpA and its cognate transporter HrpB, is identified and characterized in this study. Although all meningococcal strains harbor at least one TPS system, the
hrpA
genes display significant C-terminal sequence variation. Meningococcal genes encoding the TPS effector proteins and their transporters are closely associated and transcribed into a single mRNA. HrpA proteins are translocated across the meningococcal outer membrane by their cognate transporters HrpB and mainly released into the environment. During this process, HrpA is proteolytically processed to a mature 180-kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA remains associated with the bacteria and contributes to the interaction of meningococci with epithelial cells.
Flagella Facilitate Escape ofSalmonellafrom Oncotic Macrophages
Authors: Sano Gen-ichiro , Takada Yasunari , Goto Shinichi , Maruyama Kenta , Shindo Yutaka , Oka Kotaro , Matsui Hidenori , Matsuo Koichi
Published: September 2007

The intracellular parasiteSalmonella entericaserovar Typhimurium causes a typhoid-like systemic disease in mice. Whereas the survival ofSalmonellain phagocytes is well understood, little has been documented about the exit of intracellularSalmonellafrom host cells. Here we report that in a population of infected macrophagesSalmonellainduces “oncosis,” an irreversible progression to eukaryotic cell death characterized by swelling of the entire cell body. Oncotic macrophages (OnMφs) are terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling negative and lack actin filaments (F-actin). The plasma membrane of OnMφs filled with bacilli remains impermeable, and intracellularSalmonellabacilli move vigorously using flagella. Eventually, intracellularSalmonellabacilli intermittently exit host cells in a flagellum-dependent manner. These results suggest that induction of macrophage oncosis and intracellular accumulation of flagellated bacilli constitute a strategy wherebySalmonellaescapes from host macrophages.
Biofilm Formation-Gene Expression Relay System inEscherichia coli: Modulation of σS-Dependent Gene Expression by the CsgD Regulatory Protein via σSProtein Stabilization
Authors: Gualdi Luciana , Tagliabue Letizia , Landini Paolo
Published: September 2007

Bacteria can switch from a single-cell (planktonic) mode to a multicellular community (biofilm) mode via production of cell-cell aggregation and surface adhesion factors. In this report, we present evidence that the CsgD protein, a transcription regulator involved in biofilm formation inEscherichia coli, modulates the expression of therpoS(σS) regulon. Protein pattern analysis ofE. colicells in stationary phase shows that CsgD affects the expression of several proteins encoded by σS-dependent genes. CsgD regulation of σS-dependent genes takes place at gene transcription level, does not bypass the need forrpoS, and is abolished in anrpoS-null mutant. Consistent with these results, we find that CsgD expression leads to an increase in σSintracellular concentration. Increase in σScellular amount is mediated by CsgD-dependent transcription activation ofiraP, encoding a factor involved in σSprotein stabilization. Our results strongly suggest that the CsgD regulatory protein plays a major role as a relay between adhesion factors production and σS-dependent gene expression via σSprotein stabilization. Direct coordination between biofilm formation and expression of therpoSregulon could positively impact important biological processes, such as host colonization or response to environmental stresses.
Iteron-Binding ORF157 and FtsZ-Like ORF156 Proteins Encoded by pBtoxis Play a Role in Its Replication inBacillus thuringiensissubsp.israelensis
Authors: Tang Mujin , Bideshi Dennis K. , Park Hyun-Woo , Federici Brian A.
Published: September 2007

We recently identified a minireplicon of pBtoxis fromBacillus thuringiensissubsp.israelensisthat contained an operon encoding two novel proteins (ORF156 and ORF157), both of which are required for replication. ORF157 contains a helix-turn-helix motif and shares no homology with known plasmid replication proteins (Rep), and ORF156 contains the signature motif present in FtsZ/tubulin proteins, the latter of which are known to function in cell division and chromosome segregation. Here we show that the minimal sequence composed of four 12-bp imperfect direct repeats (iterons) in the pBtoxis minireplicon was sufficient to replicate a reporter plasmid inB. thuringiensissubsp.israelensiswhen ORF156 and ORF157 functions were provided intrans. To further investigate the roles of ORF156 and ORF157 in pBtoxis replication, six-histidine-tagged recombinant rORF156 and rORF157 proteins were purified fromEscherichia coliand used in electrophoretic mobility shift assays. Our results demonstrated that rORF157, but not rORF156, binds specifically to the pBtoxis iterons, suggesting that ORF157 functions as a Rep protein. Although rORF156 did not bind to the iteron sequence, we showed that it bound to rORF157-DNA complexes. In addition, we showed that rORF156 has GTPase activity characteristic of the FtsZ/tubulin superfamily of proteins. Taken together, these results suggest that the iterons compose the minimal replication origin (ori) of pBtoxis and that ORF157 and ORF156 are involved in the initiation of pBtoxis replication and possibly in the segregation and partitioning of this plasmid to daughter cells.
Diminished LcrV Secretion Attenuates
Yersinia pseudotuberculosis
Virulence
Authors: Bröms Jeanette E. , Francis Matthew S. , Forsberg Åke
Published: September 2007

Many gram-negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion, namely, antihost effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the translocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside bacteria, and immunomodulation via interaction with Toll-like receptor 2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting, and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation, as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.
Role for the RecBCD Recombination Pathway for
pilE
Gene Variation in Repair-Proficient
Neisseria gonorrhoeae
Authors: Hill Stuart A. , Woodward Tracy , Reger Andrew , Baker Rachel , Dinse Theresa
Published: September 2007

The role of the RecBCD recombination pathway in PilE antigenic variation in
Neisseria gonorrhoeae
is contentious and appears to be strain dependent. In this study,
N. gonorrhoeae
strain MS11
recB
mutants were assessed for recombination/repair. MS11
recB
mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth;
recB
growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9
rec
+
bacteria presenting pronounced recombination/repair defects. Consequently, MS11
recB
mutants present a more robust phenotype than the other strains that were tested. In addition, MS11
recB
mutants are also shown to be defective for
pilE/pilS
recombination. Moreover,
pilE/pilS
recombination is shown to proceed with gonococci that carry inverted
pilE
loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.
Dissection of the Caffeate Respiratory Chain in the Acetogen
Acetobacterium woodii
: Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site
Authors: Imkamp Frank , Biegel Eva , Jayamani Elamparithi , Buckel Wolfgang , Müller Volker
Published: September 2007

The anaerobic acetogenic bacterium
Acetobacterium woodii
couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of
A. woodii
catalyzes H
2
-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD
+
oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na
+
pump. These data suggest the following electron transport chain: H
2
→ ferredoxin → NAD
+
→ Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD
+
reduction catalyzed by Rnf.
Computational Prediction and Experimental Verification of the Gene Encoding the NAD+/NADP+-Dependent Succinate Semialdehyde Dehydrogenase inEscherichia coli
Authors: Fuhrer Tobias , Chen Lifeng , Sauer Uwe , Vitkup Dennis
Published: September 2007

Although NAD+-dependent succinate semialdehyde dehydrogenase activity was first described inEscherichia colimore than 25 years ago, the responsible gene has remained elusive so far. As an experimental proof of concept for a gap-filling algorithm for metabolic networks developed earlier, we demonstrate here that theE. coligeneyneIis responsible for this activity. Our biochemical results demonstrate that theyneI-encoded succinate semialdehyde dehydrogenase can use either NAD+or NADP+to oxidize succinate semialdehyde to succinate. The gene is induced by succinate semialdehyde, and expression data indicate thatyneIplays a unique physiological role in the general nitrogen metabolism ofE. coli. In particular, we demonstrate using mutant growth experiments that theyneIgene has an important, but not essential, role during growth on arginine and probably has an essential function during growth on putrescine as the nitrogen source. The NADP+-dependent succinate semialdehyde dehydrogenase activity encoded by the functional homologgabDappears to be important for nitrogen metabolism under N limitation conditions. TheyneI-encoded activity, in contrast, functions primarily as a valve to prevent toxic accumulation of succinate semialdehyde. Analysis of available genome sequences demonstrated that orthologs of bothyneIandgabDare broadly distributed across phylogenetic space.
The
Escherichia coli
Yej Transporter Is Required for the Uptake of Translation Inhibitor Microcin C
Authors: Novikova Maria , Metlitskaya Anastasia , Datsenko Kirill , Kazakov Teymur , Kazakov Alexey , Wanner Barry , Severinov Konstantin
Published: September 2007

Microcin C (McC), a peptide-nucleotide antibiotic, targets aspartyl-tRNA synthetase. By analyzing a random transposon library, we identified
Escherichia coli
mutants resistant to McC. Transposon insertions were localized to a single locus,
yejABEF
, which encodes components of a putative inner membrane ABC transporter. Analysis of site-specific mutants established that all four components of the transporter are required for McC sensitivity. Since aspartyl-tRNA synthetase in
yej
mutant extracts was fully sensitive to McC, we conclude that
yej
mutations interfere with McC uptake and that YejABEF is the only inner membrane transporter responsible for McC uptake in
E. coli
. Other substrates of YejABEF remain to be identified.
Identification of a
Vibrio furnissii
Oligopeptide Permease and Characterization of Its In Vitro Hemolytic Activity
Authors: Wu Tung-Kung , Wang Yu-Kuo , Chen Yi-Chin , Feng Jen-Min , Liu Yen-Hsi , Wang Ting-Yi
Published: September 2007

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from
Vibrio furnissii
that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a
V. furnissii hly-oppA
knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other
Vibrio
species.