bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTDuring 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

ABSTRACT

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

ABSTRACTChlamydiaspp. 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

ABSTRACT

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

ABSTRACT

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.