bluuebunny/crossref_metadata_2025 huggingface dataset:
S-Nitrosylation of Peroxiredoxin II E Promotes Peroxynitrite-Mediated Tyrosine Nitration
Authors: Romero-Puertas Maria C. , Laxa Miriam , Mattè Alessandro , Zaninotto Federica , Finkemeier Iris , Jones Alex M.E. , Perazzolli Michele , Vandelle Elodie , Dietz Karl-Josef , Delledonne Massimo
Published: December 2007

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO−), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O2  − that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO− detoxification activity of PrxII E, causing a dramatic increase of ONOO−-dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO−, indicating that the PrxII E modulation of ONOO− bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species– and NO-mediated signaling events.
Bobel-24 Activity against
Cryptosporidium parvum
in Cell Culture and in a SCID Mouse Model
Authors: Rueda Cristina , Fenoy Soledad , Simón Fernando , del Aguila Carmen
Published: December 2007

The anticryptosporidial activity of Bobel-24 (2,4,6-triiodophenol) was studied for the first time, resulting in a reduction of the in vitro growth of
Cryptosporidium
of up to 99.6%. In a SCID mouse model of chronic cryptosporidiosis, significant differences (
P
< 0.05) in oocyst shedding were observed in animals treated with 125 mg/kg/day. These results merit further investigation of Bobel-24 as a chemotherapeutic option for cryptosporidiosis.
E240V Substitution Increases Catalytic Efficiency toward Ceftazidime in a New Natural TEM-Type Extended-Spectrum β-Lactamase, TEM-149, from
Enterobacter aerogenes
and
Serratia marcescens
Clinical Isolates
Authors: Perilli Mariagrazia , Celenza Giuseppe , De Santis Francesca , Pellegrini Cristina , Forcella Chiara , Rossolini Gian Maria , Stefani Stefania , Amicosante Gianfranco
Published: December 2007

The aim of this study was to characterize a novel extended-spectrum β-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149
T182M
, in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The
bla
TEM-149
and
bla
TEM-149/T182M
genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant
Escherichia coli
HB101 by the same procedure. Both enzymes hydrolyzed all β-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149
T182M
enzymes, a reduction of the catalytic efficiency for the TEM-149
T182M
mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 β-lactamase.
Effects ofEscherichia coliLipopolysaccharide on Telithromycin Pharmacokinetics in Rats: Inhibition of Metabolism via CYP3A
Authors: Lee Joo H. , Cho Yu K. , Jung Young S. , Kim Young C. , Lee Myung G.
Published: December 2007

It has been reported that telithromycin is metabolized primarily via hepatic microsomal cytochrome P450 (CYP) 3A1/2 in rats and that the expression of hepatic and intestinal CYP3A decreases in rats pretreated withEscherichia colilipopolysaccharide (ECLPS rats; an animal model of inflammation). Thus, it is possible that the area under the plasma concentration-time curve from 0 h to infinity (AUC0-∞) of intravenous and oral telithromycin is greater for ECLPS rats than for the controls. To assess this, the pharmacokinetic parameters of telithromycin were compared after intravenous and oral administration (50 mg/kg). After intravenous administration of telithromycin, the AUC0-∞was significantly greater (by 83.4%) in ECLPS rats due to a significantly lower nonrenal clearance (by 44.5%) than in the controls. This may have been due to a significantly decreased hepatic metabolism of telithromycin in ECLPS rats. After oral administration of telithromycin, the AUC0-∞in ECLPS rats was also significantly greater (by 140%) than in the controls and the increase was considerably greater than the 83.4% increase after intravenous administration. This could have been due to a decrease in intestinal metabolism in addition to a decreased hepatic metabolism of telithromycin in ECLPS rats.
Protozoan Migration in Bent Microfluidic Channels
Authors: Wang Wei , Shor Leslie M. , LeBoeuf Eugene J. , Wikswo John P. , Taghon Gary L. , Kosson David S.
Published: December 2007

Microfluidic devices permit direct observation of microbial behavior in defined microstructured settings. Here, the swimming speed and dispersal of individual marine ciliates in straight and bent microfluidic channels were quantified. The dispersal rate and swimming speed increased with channel width, decreased with protozoan size, and was significantly impacted by the channel turning angle.
Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine
Bacillus
Species
Authors: Dick Gregory J. , Torpey Justin W. , Beveridge Terry J. , Tebo Bradley M.
Published: December 2007

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine
Bacillus
spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two
Bacillus
species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse
Bacillus
species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase.
Applicability of Phylogenetic Methods for Characterizing the Public Health Significance of Verocytotoxin-Producing
Escherichia coli
Strains
Authors: Ziebell Kim , Konczy Paulina , Yong Irene , Frost Shelley , Mascarenhas Mariola , Kropinski Andrew M. , Whittam Thomas S. , Read Susan C. , Karmali Mohamed A.
Published: December 2007

Two phylogenetic methods (multilocus sequence typing [MLST] and a multiplex PCR) were investigated to determine whether phylogenetic classification of verocytotoxin-producing
Escherichia coli
serotypes correlates with their classification into groups (seropathotypes A to E) based on their relative incidence in human disease and on their association with outbreaks and serious complications. MLST was able to separate 96% of seropathotype D and E serotypes from those that cause serious disease (seropathotypes A to C), whereas the multiplex PCR lacked this level of seropathotype discrimination.
cadDX
Operon of
Streptococcus salivarius
57.I
Authors: Chen Yi-Ywan M. , Feng C. W. , Chiu C. F. , Burne Robert A.
Published: December 2007

A CadDX system that confers resistance to Cd
2+
and Zn
2+
was identified in
Streptococcus salivarius
57.I. Unlike with other CadDX systems, the expression of the
cad
promoter was negatively regulated by CadX, and the repression was inducible by Cd
2+
and Zn
2+
, similar to what was found for CadCA systems. The lower G+C content of the
S. salivarius cadDX
genes suggests acquisition by horizontal gene transfer.
Retrospective Study of
Campylobacter
Infection in a Zoological Collection
Authors: Taema Maged M. , Bull James C. , Macgregor Shaheed K. , Flach Edmund J. , Boardman Wayne S. , Routh Andrew D.
Published: December 2007

Little is known about the epidemiology of
Campylobacter
spp. in wild animal populations. However, zoological collections can provide valuable insights. Using records from the Zoological Society of London Whipsnade Zoo compiled between 1990 and 2003, the roles of a range of biotic and abiotic factors associated with the occurrence of campylobacteriosis were investigated. The occurrence of campylobacteriosis varied widely across host taxonomic orders. Furthermore, in mammals, a combination of changes in both rainfall and temperature in the week preceding the onset of gastroenteritis were associated with isolation of
Campylobacter
from feces. In birds, there was a weak negative correlation between mean weekly rainfall and isolation of
Campylobacter
from feces. Importantly, in birds we found that the mean weekly rainfall 3 to 4 weeks before symptoms of gastroenteritis appeared was the best predictor of
Campylobacter
infection.
Campylobacter
-related gastroenteritis cases with mixed concurrent infections were positively associated with the presence of parasites (helminths and protozoans) in mammals, while in birds
Campylobacter
was associated with other concurrent bacterial infections rather than with the presence of helminths and protozoans. This study suggests that climatic elements are important factors associated with
Campylobacter
-related gastroenteritis. Further investigations are required to improve our understanding of
Campylobacter
epidemiology in captive wild animal populations.
Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product
Authors: Guglielmetti Simone , De Noni Ivano , Caracciolo Federica , Molinari Francesco , Parini Carlo , Mora Diego
Published: December 2007

Lactobacillus helveticus
MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties.
Mitochondrial
cob
and
cox1
Genes and Editing of the Corresponding mRNAs in
Dinophysis acuminata
from Narragansett Bay, with Special Reference to the Phylogenetic Position of the Genus
Dinophysis
Authors: Zhang Huan , Bhattacharya Debashish , Maranda Lucie , Lin Senjie
Published: December 2007

Dinophysis acuminata
cells were isolated from Narragansett Bay water samples in June 2005 using flow cytometry. Dinoflagellate-specific PCR primers were used to isolate small-subunit rRNA (18S rRNA), mitochondrial cytochrome
b
(
cob
), and cytochrome
c
oxidase I (
cox1
) genes and the encoded cDNAs. Maximum-likelihood analysis of a concatenated data set of ribosomal DNA and cDNA sequences of
cob
and
cox1
showed that
D. acuminata
was sister to Gonyaulacoids, but without strong bootstrap support. The approximately unbiased test could not reject alternative positions of
D. acuminata
. To gain better resolution, mRNA editing of
cob
and
cox1
was inferred for
D. acuminata
and 13 other dinoflagellate species. The location and type of editing as well as the distribution pattern in
D. acuminata
were generally similar to those in other dinoflagellates except for two edited sites that are unique to this species. Bayesian analyses of a matrix that recorded the location and type of editing, and of a matrix that included the protein sequences of COB and COX1 with the editing data yielded tree topologies similar to the three-gene tree but again failed to resolve the phylogenetic position of
D. acuminata
. However, the density of edited sites in the
D. acuminata
mitochondrial genes, consistent with phylogenetic trees, indicated that
Dinophysis
is a derived dinoflagellate lineage, diverging after other lineages such as
Oxyrrhis
,
Amphidinium
, and
Symbiodinium
. We demonstrate that dinoflagellate-specific PCR coupled with flow cytometry can be a useful tool to analyze genes and their transcripts from a natural dinoflagellate population.
Detection of Plant-Modulated Alterations in Antifungal Gene Expression in
Pseudomonas fluorescens
CHA0 on Roots by Flow Cytometry
Authors: de Werra Patrice , Baehler Eric , Huser Aurélie , Keel Christoph , Maurhofer Monika
Published: December 2007

The biocontrol activity of the root-colonizing
Pseudomonas fluorescens
strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the
phlA
and
prnA
genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of
phlA
-
gfp
and
prnA
-
gfp
in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the
gfp
reporters, the average gene expression per bacterial cell could be calculated. Levels of
phlA
and
prnA
expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered
phlA
expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.
Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events
Authors: Rizzi Aurora , Pontiroli Alessandra , Brusetti Lorenzo , Borin Sara , Sorlini Claudia , Abruzzese Alessandro , Sacchi Gian Attilio , Vogel Timothy M. , Simonet Pascal , Bazzicalupo Marco , Nielsen Kaare Magne , Monier Jean-Michel , Daffonchio Daniele
Published: December 2007

A strategy is described that enables the in situ detection of natural transformation in
Acinetobacter baylyi
BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.
Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in
Escherichia coli
Authors: Gursinsky Torsten , Gröbe Daniel , Schierhorn Angelika , Jäger Jana , Andreesen Jan R. , Söhling Brigitte
Published: December 2007

Selenoprotein synthesis in
Escherichia coli
strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe
Eubacterium acidaminophilum
in
E. coli
was studied. Incorporation of
75
Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an
E. coli
wild-type strain and was fully absent in an
E. coli
SelB mutant. Selenoprotein synthesis, however, was strongly increased if
selB
and
selC
(tRNA
Sec
) from
E. acidaminophilum
were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the
E. coli
SECIS element. However, mutations in these structures strongly reduced the amount of
75
Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using
gst-lacZ
translational fusions. In the presence of
selB
and
selC
from
E. acidaminophilum
, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in
Desulfomicrobium baculatum
(
hydV
),
Treponema denticola
(
selD
), and
Campylobacter jejuni
(
selW
-like gene) was also considerably enhanced in the presence of
E. acidaminophilum selB
and
selC.
This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in
E. coli
.
Genetic Rearrangement Strategy for Optimizing the Dibenzothiophene Biodesulfurization Pathway in
Rhodococcus erythropolis
Authors: Li Guo-qiang , Li Shan-shan , Zhang Ming-lu , Wang Jun , Zhu Lin , Liang Feng-lai , Liu Ru-lin , Ma Ting
Published: December 2007

Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by enzymes DszC, DszA, and DszB, which are encoded by the operon
dszABC
and contribute to the conversion in tandem. We investigated the expression characteristics of the
dsz
operon. Our results revealed that the levels of transcription and translation of
dszA
,
dszB
, and
dszC
decreased according to the positions of the genes in the
dsz
operon. Furthermore, the translation of
dszB
was repressed by an overlapping structure in the
dsz
operon. In order to get better and steady expression of the Dsz enzymes and optimize the metabolic flux of DBT, we rearranged the
dsz
operon according to the catalytic capabilities of the Dsz enzymes and expressed the rearranged
dsz
operon,
dszBCA
, in
Rhodococcus erythropolis
. After rearrangement, the ratio of
dszA
,
dszB
, and
dszC
mRNAs in the cells was changed, from 11:3.3:1 to 1:16:5. Western blot analysis revealed that the levels of expression of
dszB
and
dszC
had been enhanced but that the expression of
dszA
had decreased. The desulfurization activity of resting cells prepared from
R. erythropolis
DRB, which carried the rearranged
dsz
operon, was about 12-fold higher than that of resting cells of
R. erythropolis
DRA, which carried the original operon in a similarly constructed vector.
Marker Removal in Staphylococci via Cre Recombinase and Different
lox
Sites
Authors: Leibig Martina , Krismer Bernhard , Kolb Martina , Friede Alexandra , Götz Friedrich , Bertram Ralph
Published: December 2007

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-
lox
setting was established that allowed the efficient removal of resistance genes from the genomes of
Staphylococcus carnosus
and
S. aureus
. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant
lox
sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed
cre
expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci.
A
Legionella pneumophila
Peptidyl-Prolyl
cis
-
trans
Isomerase Present in Culture Supernatants Is Necessary for Optimal Growth at Low Temperatures
Authors: Söderberg Maria A. , Cianciotto Nicholas P.
Published: December 2007

Several
Legionella pneumophila
proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking
ppiB
exhibited reduced growth at 17°C. Since PpiB lacked a signal sequence and was present in 17°C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.
The
Escherichia coli
Efflux Pump TolC Promotes Aggregation of Enteroaggregative
E. coli
042
Authors: Imuta Naoko , Nishi Junichiro , Tokuda Koichi , Fujiyama Rika , Manago Kunihiro , Iwashita Mayumi , Sarantuya Jav , Kawano Yoshifumi
Published: December 2007

Enteroaggregative
Escherichia coli
(EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an
E. coli
efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its
tolC
mutant (042
tolC
). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042
tolC
. In a microtiter plate assay, there were significantly more planktonic cells for 042
tolC
than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042
tolC
than for the wild type. In a HEp-2 cell adherence test, 042
tolC
showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with
tolC
. In a transwell assay, planktonic cells of 042
tolC
decreased when cocultured with the wild type or the complement, while precipitated cells of 042
tolC
increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.
Influence of dTMP on the Phenotypic Appearance and Intracellular Persistence of
Staphylococcus aureus
Authors: Zander Johannes , Besier Silke , Saum Stephan H. , Dehghani Faramarz , Loitsch Stefan , Brade Volker , Wichelhaus Thomas A.
Published: December 2007

Thymidine-dependent small-colony variants (SCVs) of
Staphylococcus aureus
are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of
S. aureus
SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of
S. aureus
. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of
S. aureus
.
Novel Adhesin from
Pasteurella multocida
That Binds to the Integrin-Binding Fibronectin FnIII
9-10
Repeats
Authors: Mullen Lisa M. , Nair Sean P. , Ward John M. , Rycroft Andrew N. , Williams Rachel J. , Robertson Giles , Mordan Nicky J. , Henderson Brian
Published: December 2007

Phage display screening with fragmented genomic DNA from the animal pathogen
Pasteurella multocida
has identified a gene encoding a putative fibronectin binding protein (19). Homologues of this gene (PM1665) are found in all other sequenced members of the
Pasteurellaceae
. Gene PM1665 has been cloned, and the protein has been expressed. Recombinant PM1665 protein binds to both soluble and immobilized fibronectin and is unique in that it interacts with the integrin-binding fibronectin type III (FnIII) repeats FnIII
9-10
and not, as is the case for almost all other fibronectin adhesins, to the N-terminal type I repeats. Surface plasmon resonance analysis revealed a complex binding mechanism with a

K
D

(equilibrium dissociation constant) of 150 nM ± 70 nM. Bioinformatics analysis suggests that the PM1665 protein contains two helix-hairpin-helix (HhH) motifs, and truncation mutation studies have identified the binding site in the protein as a combination of these two HhH motifs in conjunction with a conserved amino acid motif, VNINTA. We have shown that the PM1665 protein is on the cell surface and that binding of
P. multocida
to fibronectin is almost completely inhibited by anti-PM1665 antiserum. These results support the hypothesis that the PM1665 protein is a member of a new family of fibronectin binding adhesins that are important in the adhesion of
P. multocida
to fibronectin.
The Monofunctional Glycosyltransferase ofEscherichia coliLocalizes to the Cell Division Site and Interacts with Penicillin-Binding Protein 3, FtsW, and FtsN
Authors: Derouaux Adeline , Wolf Benoît , Fraipont Claudine , Breukink Eefjan , Nguyen-Distèche Martine , Terrak Mohammed
Published: December 2007

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site ofEscherichia colicells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.
Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry
Authors: Vuong Phu , Bennion Drew , Mantei Jeremy , Frost Danielle , Misra Rajeev
Published: December 2007

In
Escherichia coli
, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for β-barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or
yfgL
-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the
yfgL
background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL.
The Type III Toxins of
Pseudomonas aeruginosa
Disrupt Epithelial Barrier Function
Authors: Soong Grace , Parker Dane , Magargee Mariah , Prince Alice S.
Published: December 2007

The type III secreted toxins of
Pseudomonas aeruginosa
are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to ΔSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance
P. aeruginosa
's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.
α-Tocopherol Is Essential for Acquired Chill-Light Tolerance in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Yang Yang , Yin Chuntao , Li Weizhi , Xu Xudong
Published: December 2007

Unlike
Escherichia coli
, the cyanobacterium
Synechocystis
sp. strain PCC 6803 is insensitive to chill (5°C) in the dark but rapidly losses viability when exposed to chill in the light (100 μmol photons m
−2
s
−1
). Preconditioning at a low temperature (15°C) greatly enhances the chill-light tolerance of
Synechocystis
sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of α-tocopherol after exposure to chill-light stress. Mutants unable to synthesize α-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P

petE

controlled the level of α-tocopherol and ACLT. We conclude that α-tocopherol is essential for ACLT of
Synechocystis
sp. strain PCC 6803. The role of α-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.
Unusual Properties of Catalase A (KatA) of
Pseudomonas aeruginosa
PA14 Are Associated with Its Biofilm Peroxide Resistance
Authors: Shin Dong-Ho , Choi Young-Seok , Cho You-Hee
Published: December 2007

Pseudomonas aeruginosa
is a ubiquitous environmental bacterium whose major catalase (KatA) is highly stable, extracellularly present, and required for full virulence as well as for peroxide resistance in planktonic and biofilm states. Here, we dismantled the function of
P. aeruginosa
KatA (KatA
Pa
) by comparing its properties with those of two evolutionarily related (clade 3 monofunctional) catalases from
Bacillus subtilis
(KatA
Bs
) and
Streptomyces coelicolor
(CatA
Sc
). We switched the coding region for KatA
Pa
with those for KatA
Bs
and CatA
Sc
, expressed the catalases under the potential
katA
-regulatory elements in a
P. aeruginosa
PA14
katA
mutant, and verified their comparable protein levels by Western blot analysis. The activities of KatA
Bs
and CatA
Sc
, however, were less than 40% of the KatA
Pa
activity, suggestive of the difference in intrinsic catalatic activity or efficiency for posttranslational activity modulation in
P. aeruginosa
. Furthermore, KatA
Bs
and CatA
Sc
were relatively susceptible to proteinase K, whereas KatA
Pa
was highly stable upon proteinase K treatment. As well, KatA
Bs
and CatA
Sc
were undetectable in the extracellular milieu. Nevertheless,
katA
Bs
and
catA
Sc
fully rescued the peroxide sensitivity and osmosensitivity of the
katA
mutant, respectively. Both catalase genes rescued the attenuated virulence of the
katA
mutant in mouse acute infection and
Drosophila melanogaster
models. However, the peroxide susceptibility of the
katA
mutant in a biofilm growth state was rescued by neither
katA
Bs
nor
catA
Sc
. Based on these results, we propose that the
P. aeruginosa
KatA is highly stable compared to the two major catalases from gram-positive bacteria and that its unique properties involving metastability and extracellular presence may contribute to the peroxide resistance of
P. aeruginosa
biofilm and presumably to chronic infections.
3-Hydroxypropionyl-Coenzyme A Synthetase from
Metallosphaera sedula
, an Enzyme Involved in Autotrophic CO
2
Fixation
Authors: Alber Birgit E. , Kung Johannes W. , Fuchs Georg
Published: December 2007

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO
2
fixation pathway for the thermoacidophilic crenarchaeon
Metallosphaera sedula
. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min
−1
mg protein
−1
. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP
i
. The enzyme was purified 95-fold to a specific activity of 18 μmol min
−1
mg protein
−1
from autotrophically grown
M. sedula
cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of
Sulfolobus tokodaii
; similar genes were found in
S. solfataricus
and
S. acidocaldarius
. The gene was heterologously expressed in
Escherichia coli
, and the His-tagged protein was purified. Both the native enzyme from
M. sedula
and the recombinant enzyme from
S. tokodaii
not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown
M. sedula
, supporting its proposed role during CO
2
fixation in this archaeon and possibly other members of the
Sulfolobaceae
family.
Differential Role of Ferritins in Iron Metabolism and Virulence of the Plant-Pathogenic BacteriumErwinia chrysanthemi3937
Authors: Boughammoura Aïda , Matzanke Berthold F. , Böttger Lars , Reverchon Sylvie , Lesuisse Emmanuel , Expert Dominique , Franza Thierry
Published: December 2007

During infection, the phytopathogenic enterobacteriumErwinia chrysanthemihas to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. TheE. chrysanthemigenome possesses two loci that could be devoted to iron storage: thebfrgene, encoding a heme-containing bacterioferritin, and theftnAgene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike thebfrmutant, theftnAmutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, thebfr ftnAmutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in theftnAandbfr ftnAmutants. We found thatftnAgene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB.bfrgene expression is induced at the stationary phase of growth. The σStranscriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence ofE. chrysanthemidepending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.
Structural and Biological Characterization of a Capsular Polysaccharide Produced by
Staphylococcus haemolyticus
Authors: Flahaut Sigrid , Vinogradov Evgeny , Kelley Kathryn A. , Brennan Shannon , Hiramatsu Keiichi , Lee Jean C.
Published: December 2007

The DNA sequence of the genome of
Staphylococcus haemolyticus
JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (

capABCDEFG
Sh

) showed ≥57% similarity with the
Staphylococcus aureus cap5
or
cap8
locus. However, the

capHIJKLM
Sh

genes are unique to
S. haemolyticus
and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted
cap
region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid.
S. haemolyticus cap
genes were amplified from four of seven clinical isolates of
S. haemolyticus
from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from
S. haemolyticus
strain JCSC1435 showed a trisaccharide repeating unit: −3-α-
l
-FucNAc-3-(2-NAc-4-
N
-Asp-2,4,6-trideoxy-β-
d
-Glc)-4-α-
d
-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an
N
-acyl substituent.
Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis
Authors: Betts H. J. , Twiggs L. E. , Sal M. S. , Wyrick P. B. , Fields K. A.
Published: December 2007

Chlamydiaspp. express a functional type III secretion system (T3SS) necessary for pathogenesis and intracellular growth. However, certain essential components of the secretion apparatus have diverged to such a degree as to preclude their identification by standard homology searches of primary protein sequences. One example is the needle subunit protein. Electron micrographs indicate that chlamydiae possess needle filaments, and yet database searches fail to identify a SctF homologue. We used a bioinformatics approach to identify a likely needle subunit protein forChlamydia. Experimental evidence indicates that this protein, designated CdsF, has properties consistent with it being the major needle subunit protein. CdsF is concentrated in the outer membrane of elementary bodies and is surface exposed as a component of an extracellular needle-like projection. During infection CdsF is detectible by indirect immunofluorescence in the inclusion membrane with a punctuate distribution adjacent to membrane-associated reticulate bodies. Biochemical cross-linking studies revealed that, like other SctF proteins, CdsF is able to polymerize into multisubunit complexes. Furthermore, we identified two chaperones for CdsF, termed CdsE and CdsG, which have many characteristics of thePseudomonasspp. needle chaperones PscE and PscG, respectively. In aggregate, our data are consistent with CdsF representing at least one component of the extendedChlamydiaT3SS injectisome. The identification of this secretion system component is essential for studies involving ectopic reconstitution of theChlamydiaT3SS. Moreover, we anticipate that CdsF could serve as an efficacious target for anti-Chlamydianeutralizing antibodies.
Regulation of Circadian Clock Gene Expression by Phosphorylation States of KaiC in Cyanobacteria
Authors: Murayama Yoriko , Oyama Tokitaka , Kondo Takao
Published: December 2007

Three clock proteins—KaiA, KaiB, and KaiC—have been identified as essential components of the circadian oscillator in cyanobacteria, and Kai-based chemical oscillation is thought to be the basic circadian timing mechanism in
Synechococcus elongatus
PCC 7942. Transcription and translation of
kaiBC
in cyanobacterial cells was quantitatively studied to elucidate how these processes are coupled to the chemical oscillator using a strain in which circadian oscillation is under the control of IPTG (isopropyl-β-
d
-thiogalactopyranoside). The kinetics of repression of
kaiBC
promoter triggered by IPTG allowed estimation of transient response at 10 h. This response time is suitable for cyanobacterial transcription and/or translation to match with the Kai-based oscillator. Interestingly,
kaiBC
promoter activity and KaiC phosphorylation showed robust circadian rhythms, whereas
trc
promoter-driven
kaiBC
mRNA levels and KaiC accumulation were almost arrhythmic. These results indicate that cyanobacterial circadian rhythms can be generated even if
kaiBC
expression is constitutive. Moreover, there was a positive correlation between activation of the
kaiBC
promoter and an increase in the KaiC phosphorylation ratio in three rhythmic conditions. Based on these observations, it is likely that the KaiC phosphorylation ratio is the main factor in the activation of
kaiBC
promoter. Finally, we quantitatively compared the threshold level of phosphorylated KaiC for the repression or derepression of
kaiBC
promoter and found that this parameter is an important factor in repressing the
kaiBC
promoter.
Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by
Cupriavidus necator
JMP134
Authors: Belchik Sara Mae , Xun Luying
Published: December 2007

The
tcpRXABCYD
operon of
Cupriavidus necator
JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH
2
)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH
2
, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (
tcpX
and
tcpB
) were cloned, overexpressed, and purified in
Escherichia coli
. TcpX was purified as a C-terminal His tag fusion (TcpX
H
) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX
H
had no activity toward NADPH or riboflavin. Coupling of TcpX
H
and TcpA demonstrated that TcpX
H
provided FADH
2
for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX
H
in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.
PflI, a Protein Involved in Flagellar Positioning in
Caulobacter crescentus
Authors: Obuchowski Pamela L. , Jacobs-Wagner Christine
Published: December 2007

The bacterial flagellum is important for motility and adaptation to environmental niches. The sequence of events required for the synthesis of the flagellar apparatus has been extensively studied, yet the events that dictate where the flagellum is placed at the onset of flagellar biosynthesis remain largely unknown. We addressed this question for alphaproteobacteria by using the polarly flagellated alphaproteobacterium
Caulobacter crescentus
as an experimental model system. To identify candidates for a role in flagellar placement, we searched all available alphaproteobacterial genomes for genes of unknown function that cluster with early flagellar genes and that are present in polarly flagellated alphaproteobacteria while being absent in alphaproteobacteria with other flagellation patterns. From this in silico screen, we identified
pflI
. Loss of PflI function in
C. crescentus
results in an abnormally high frequency of cells with a randomly placed flagellum, while other aspects of cell polarization remain normal. In a wild-type background, a fusion of green fluorescent protein (GFP) and PflI localizes to the pole where the flagellum develops. This polar localization is independent of the flagellar protein FliF, whose oligomerization into the MS ring is thought to define the site of flagellar synthesis, suggesting that PflI acts before or independently of this event. Overproduction of PflI-GFP often leads to ectopic localization at the wrong, stalked pole. This is accompanied by a high frequency of flagellum formation at this ectopic site, suggesting that the location of PflI is a sufficient marker for a site for flagellar assembly.