bluuebunny/crossref_metadata_2025 huggingface dataset:
Gateway-Based Destination Vectors for Functional Analyses of Bacterial ORFeomes: Application to the Min System inBrucella abortus
Authors: Hallez Régis , Letesson Jean-Jacques , Vandenhaute Jean , De Bolle Xavier
Published: December 2006

Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscillating localization of MinD inBrucella abortus.
Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture
Authors: Shimamura Munetaka , Nishiyama Takashi , Shigetomo Hiroyuki , Toyomoto Takeshi , Kawahara Yuka , Furukawa Kenji , Fujii Takao
Published: December 2006

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome
c
as an electron acceptor, the

V
max

and

K
m

for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO,
hzoA
and
hzoB
, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two
hao
genes found by sequencing the genome of “
Candidatus
Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.
Mpt5p, a Stress Tolerance- and Lifespan-Promoting PUF Protein in
Saccharomyces cerevisiae
, Acts Upstream of the Cell Wall Integrity Pathway
Authors: Stewart Mark S. , Krause Sue Ann , McGhie Josephine , Gray Joseph V.
Published: December 2006

Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One
Saccharomyces cerevisiae
PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that
mpt5Δ
mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway:
mpt5Δ
mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target,
LRG1
, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling,
LRG1
mRNA contains a consensus Mpt5p-binding site in its putative 3′ untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of
mpt5Δ
mutants, and
LRG1
mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.
Temporal and Spatial Control of
HGC1
Expression Results in Hgc1 Localization to the Apical Cells of Hyphae in
Candida albicans
Authors: Wang Allen , Lane Shelley , Tian Zhen , Sharon Amir , Hazan Idit , Liu Haoping
Published: December 2006

The human fungal pathogen
Candida albicans
can undergo a morphological transition from a unicellular yeast growth form to a multicellular hyphal growth form. During hyphal growth, cell division is asymmetric. Only the apical cell divides, whereas subapical cells remain in G
1
, and cell surface growth is highly restricted to the tip of the apical cell. Hgc1, a hypha-specific, G
1
cyclin-like protein, is essential for hyphal development. Here, we report, using indirect immunofluorescence, that Hgc1 is preferentially localized to the dividing apical cells of hyphae. Hgc1 protein is rapidly degraded in a cell cycle-independent manner, and the protein turnover likely occurs in both the apical and the subapical cells of hyphae. In addition to rapid protein turnover, the
HGC1
transcript is also dynamically regulated during cell cycle progression in hyphal growth. It is induced upon germ tube formation in early G
1
; the transcript level is reduced during the G
1
/S transition and peaks again around the G
2
/M phase in the subsequent cell cycles. Transcription from the
HGC1
promoter is essential for its apical cell localization, as Hgc1 no longer exhibits preferential apical localization when expressed under the
MAL2
promoter. Using fluorescence in situ hybridization, the
HGC1
transcript is detected only in the apical cells of hyphae, suggesting that
HGC1
is transcribed in the apical cell. Therefore, the preferential localization of Hgc1 to the apical cells of hyphae results from the dynamic temporal and spatial control of
HGC1
expression.
FtcR Is a New Master Regulator of the Flagellar System ofBrucella melitensis16M with Homologs inRhizobiaceae
Authors: Léonard S. , Ferooz J. , Haine V. , Danese I. , Fretin D. , Tibor A. , de Walque S. , De Bolle X. , Letesson J.-J.
Published: December 2006

The flagellar regulon ofBrucella melitensis16M contains 31 genes clustered in three loci on the small chromosome. These genes encode a polar sheathed flagellum that is transiently expressed during vegetative growth and required for persistent infection in a mouse model. By following the expression of three flagellar genes (fliF,flgE, andfliC, corresponding to the MS ring, hook, and filament monomer, respectively), we identified a new regulator gene,ftcR(flagellartwo-componentregulator). Inactivation offtcRled to a decrease in flagellar gene expression and to impairedBrucellavirulence. FtcR has a two-component response regulator domain as well a DNA binding domain and is encoded in the first flagellar locus ofB. melitensis. Both theftcRsequence and its genomic context are conserved in other related α-proteobacteria. During vegetative growth in rich medium,ftcRexpression showed a peak during the early exponential phase that paralleledfliFgene expression. VjbR, a quorum-sensing regulator of the LuxR family, was previously found to controlfliFandflgEgene expression. Here, we provide some new elements suggesting that the effect of VjbR on these flagellar genes is mediated by FtcR. We found thatftcRexpression is partially under the control of VjbR and that the expression intransofftcRin avjbRmutant restored the production of the hook protein (FlgE). Finally, FtcR binds directly to the upstream region of thefliFgene. Therefore, our data support the role of FtcR as a flagellar master regulator inB. melitensisand perhaps in other related α-proteobacteria.
Characterization of the
Moraxella catarrhalis
Opa-Like Protein, OlpA, Reveals a Phylogenetically Conserved Family of Outer Membrane Proteins
Authors: Brooks Michael J. , Laurence Cassie A. , Hansen Eric J. , Gray-Owen Scott D.
Published: December 2006

Moraxella catarrhalis
is a human-restricted pathogen that can cause respiratory tract infections. In this study, we identified a previously uncharacterized 24-kDa outer membrane protein with a high degree of similarity to
Neisseria
Opa protein adhesins, with a predicted β-barrel structure consisting of eight antiparallel β-sheets with four surface-exposed loops. In striking contrast to the antigenically variable Opa proteins, the
M. catarrhalis
Opa-like protein (OlpA) is highly conserved and constitutively expressed, with 25 of 27 strains corresponding to a single variant. Protease treatment of intact bacteria and isolation of outer membrane vesicles confirm that the protein is surface exposed yet does not bind host cellular receptors recognized by neisserial Opa proteins. Genome-based analyses indicate that OlpA and Opa derive from a conserved family of proteins shared by a broad array of gram-negative bacteria.
Oxygen Reactivity of Both Respiratory Oxidases in
Campylobacter jejuni
: the
cydAB
Genes Encode a Cyanide-Resistant, Low-Affinity Oxidase That Is Not of the Cytochrome
bd
Type
Authors: Jackson Rachel J. , Elvers Karen T. , Lee Lucy J. , Gidley Mark D. , Wainwright Laura M. , Lightfoot James , Park Simon F. , Poole Robert K.
Published: December 2006

The microaerophilic bacterium
Campylobacter jejuni
is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (
cydAB
) apparently encoding a cytochrome
bd
-like oxidase homologous to oxidases in
Escherichia coli
and
Azotobacter vinelandii
. However,
C. jejuni
cells lacked all spectral signals characteristic of the high-spin hemes
b
and
d
of these oxidases. Mutation of the
cydAB
operon of
C. jejuni
did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that
C. jejuni
CydAB be renamed CioAB (
c
yanide-
i
nsensitive
o
xidase), as in
Pseudomonas aeruginosa
. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (

K
m

= 0.8 μM) and a
V
max
of >20 nmol/mg/s. Expression of
cioAB
was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative,
ccoNOQP
-encoded cyanide-sensitive oxidase, expected to encode a cytochrome
cb′
-type enzyme, plays a major role in the microaerobic respiration of
C. jejuni
, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a

K
m

value of 40 nM and a
V
max
of 6 to 9 nmol/mg/s. Low-temperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.
Genome Sequence of Avery's Virulent Serotype 2 Strain D39 ofStreptococcus pneumoniaeand Comparison with That of Unencapsulated Laboratory Strain R6
Authors: Lanie Joel A. , Ng Wai-Leung , Kazmierczak Krystyna M. , Andrzejewski Tiffany M. , Davidsen Tanja M. , Wayne Kyle J. , Tettelin Hervé , Glass John I. , Winkler Malcolm E.
Published: December 2006

Streptococcus pneumoniae(pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only twoS. pneumoniaestrains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.
A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate
Authors: Cámara Beatriz , Bielecki Piotr , Kaminski Filip , dos Santos Vitor Martins , Plumeier Iris , Nikodem Patricia , Pieper Dietmar H.
Published: December 2006

Pseudomonassp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, withsalA, encoding a salicylate 1-hydroxylase, being clustered withsalCDgenes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typicalcatRBCAarrangement of genes of the catechol branch of the 3-oxoadipate pathway inPseudomonasstrains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by thecatgene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by thesalcluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of thesalcluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of thesalgene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catecholorthocleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.
Loss of a Universal tRNA Feature
Authors: Wang Chunxia , Sobral Bruno W. , Williams Kelly P.
Published: December 2006

tRNA
His
has thus far always been found with one of the most distinctive of tRNA
features, an extra 5′ nucleotide that is usually a guanylate.
tRNA
His
genes in a disjoint alphaproteobacterial group
comprising the
Rhizobiales
,
Rhodobacterales
,
Caulobacterales
,
Parvularculales
, and
Pelagibacter
generally fail to encode this extra guanylate,
unlike those of other alphaproteobacteria and bacteria in general.
Rather than adding an extra 5′ guanylate posttranscriptionally
as eukaryotes do, evidence is presented here that two of these species,
Sinorhizobium meliloti
and
Caulobacter crescentus
,
simply lack any extra nucleotide on tRNA
His
. This loss
correlates with changes at the 3′ end sequence of
tRNA
His
and at many sites in histidyl-tRNA synthetase that
might be expected to affect tRNA
His
recognition, in the
flipping loop, the insertion domain, the anticodon-binding domain, and
the motif 2 loop. The altered tRNA charging system may have affected
other tRNA charging systems in these bacteria; for example, a site in
tRNA
Glu
sequences was found to covary with
tRNA
His
among
alphaproteobacteria.
Influence of Wall Teichoic Acid on Lysozyme Resistance inStaphylococcus aureus
Authors: Bera Agnieszka , Biswas Raja , Herbert Silvia , Kulauzovic Emir , Weidenmaier Christopher , Peschel Andreas , Götz Friedrich
Published: December 2006

Staphylococcus aureuspeptidoglycan (PG) is completely resistant to the hydrolytic activity of lysozyme. Here we show that modifications in PG by O acetylation, wall teichoic acid, and a high degree of cross-linking contribute to this resistance.
Role of FtsEX in Cell Division of
Escherichia coli
: Viability of
ftsEX
Mutants Is Dependent on Functional SufI or High Osmotic Strength
Authors: Reddy Manjula
Published: December 2006

In
Escherichia coli
, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that
ftsEX
null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of
ftsEX
mutants.
ftsEX
deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the
ftsZ84
allele was a weak suppressor of
ftsEX
. The viability of
ftsEX
mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.
Identification of IspC, an 86-Kilodalton Protein Target of Humoral Immune Response to Infection with
Listeria monocytogenes
Serotype 4b, as a Novel Surface Autolysin
Authors: Wang Linru , Lin Min
Published: December 2006

We identified and biochemically characterized a novel surface-localized autolysin from
Listeria monocytogenes
serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in
Escherichia coli
, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium
Micrococcus lysodeikticus
and the gram-negative bacterium
E. coli
but also that of the IspC-producing strain of
L. monocytogenes
serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.
SRP19 Is a Dispensable Component of the Signal Recognition Particle in
Archaea
Authors: Yurist Sophie , Dahan Idit , Eichler Jerry
Published: December 2006

In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in
Archaea
. Accordingly, the
Haloferax volcanii SRP19
gene was deleted. Although normally transcribed at a level comparable to that of the essential
SRP54
gene,
SRP19
deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.
Complete Genome Sequence of
Haemophilus somnus
(
Histophilus somni
) Strain 129Pt and Comparison to
Haemophilus ducreyi
35000HP and
Haemophilus influenzae
Rd
Authors: Challacombe Jean F. , Duncan A. J. , Brettin Thomas S. , Bruce David , Chertkov Olga , Detter J. Chris , Han Cliff S. , Misra Monica , Richardson Paul , Tapia Roxanne , Thayer Nina , Xie Gary , Inzana Thomas J.
Published: December 2006

Haemophilus somnus
can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of
H. somnus
are a significant cause of systemic disease in cattle. We report the genome sequence of
H. somnus
129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of
H. somnus
129Pt,
Haemophilus influenzae
Rd, and
Haemophilus ducreyi
35000HP. We found unique genes in
H. somnus
129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of
H. somnus
129Pt,
H. influenzae
Rd, and
H. ducreyi
35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between
H. somnus
129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against
H. somnus
disease.
The Crystal Structure of Rv0813c from
Mycobacterium tuberculosis
Reveals a New Family of Fatty Acid-Binding Protein-Like Proteins in Bacteria
Authors: Shepard William , Haouz Ahmed , Graña Martin , Buschiazzo Alejandro , Betton Jean-Michel , Cole Stewart T. , Alzari Pedro M.
Published: December 2006

The gene Rv0813c from
Mycobacterium tuberculosis
, which codes for a hypothetical protein of unknown function, is conserved within the order
Actinomycetales
but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded β-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol.
The
Sphingomonas
Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole
Authors: Shintani Masaki , Urata Masaaki , Inoue Kengo , Eto Kaori , Habe Hiroshi , Omori Toshio , Yamane Hisakazu , Nojiri Hideaki
Published: December 2006

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from
Sphingomonas
sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading
Novosphingobium aromaticivorans
strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (
car
-I and
car
-II), and genes for electron transfer components of initial oxygenase for carbazole (
fdxI
,
fdrI
, and
fdrII
). Putative genes were identified for the degradation of anthranilate (
and
), catechol (
cat
), 2-hydroxypenta-2,4-dienoate (
carDFE
), dibenzofuran/fluorene (
dbf/fln
), protocatechuate (
lig
), and phthalate (
oph
). It appears that pCAR3 may carry clustered genes (
car
-I,
car
-II,
fdxI
,
fdrI
,
fdrII
,
and
, and
cat
) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of
car
-I,
car
-II, and
cat
genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from
car
-I,
car-
II,
repA
,
parA
,
traI
, and
traD
genes indicated that several
Sphingomonas
carbazole degraders have DNA regions similar to parts of pCAR3.
A Suppressor of Cell Death Caused by the Loss of σ
E
Downregulates Extracytoplasmic Stress Responses and Outer Membrane Vesicle Production in
Escherichia coli
Authors: Button Julie E. , Silhavy Thomas J. , Ruiz Natividad
Published: December 2006

When envelope biogenesis is compromised or damage to envelope components occurs, bacteria trigger signaling cascades, which lead to the production of proteins that combat such extracytoplasmic stresses. In
Escherichia coli
, there are three pathways known to deal with envelope stresses: the Bae, Cpx, and σ
E
responses. Although the effectors of the Bae and Cpx responses are not essential in
E. coli
, the effector of the σ
E
response, the sigma factor RpoE (σ
E
), is essential for viability. However, mutations that suppress the lethality of an
rpoE
-null allele can be easily obtained, and here we describe how we have isolated at least four classes of these suppressors. We present the first description of one such suppressor class, loss-of-function mutations in
ydcQ
, a gene encoding a putative DNA-binding protein. In wild-type
rpoE

+

strains,
ydcQ
mutants have two distinct phenotypes: extracytoplasmic stress responses are significantly downregulated, and the production of outer membrane vesicles is severely reduced. We present a model in which σ
E
is not essential per se but, rather, we propose that
rpoE
mutant cells die, possibly because they overreact to the absence of this σ factor by triggering a cell death signal.
Porin Activity of
Anaplasma phagocytophilum
Outer Membrane Fraction and Purified P44
Authors: Huang Haibin , Wang Xueqi , Kikuchi Takane , Kumagai Yumi , Rikihisa Yasuko
Published: December 2006

Anaplasma phagocytophilum
, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus,
A. phagocytophilum
needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of
A. phagocytophilum
has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of
l
-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of
A. phagocytophilum
. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel β-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100
p44
paralogs and go through extensive antigenic variation. The 16-transmembrane-domain β-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of
A. phagocytophilum
is incomplete and requires the exogenous acquisition of
l
-glutamine or
l
-glutamate for function. Efficient diffusion of
l
-glutamine across the outer membrane suggests that the porin feeds the
Anaplasma
TCA cycle and that the relatively large pore size provides
Anaplasma
with the necessary metabolic intermediates from the host cytoplasm.
The
Pseudomonas
Quorum-Sensing Regulator RsaL Belongs to the Tetrahelical Superclass of H-T-H Proteins
Authors: Rampioni Giordano , Polticelli Fabio , Bertani Iris , Righetti Karima , Venturi Vittorio , Zennaro Elisabetta , Leoni Livia
Published: December 2006

In the opportunistic human pathogen
Pseudomonas aeruginosa
, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of
lasI
, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the
N
-terminal domain of the lambda
c
I repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the
lasI
promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.
Altered Levels ofSalmonellaDNA Adenine Methylase Are Associated with Defects in Gene Expression, Motility, Flagellar Synthesis, and Bile Resistance in the Pathogenic Strain 14028 but Not in the Laboratory Strain LT2
Authors: Badie Golnaz , Heithoff Douglas M. , Sinsheimer Robert L. , Mahan Michael J.
Published: December 2006

Comparative genomic analysis has revealed limited strain diversity betweenSalmonellapathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels ofSalmonellaDNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in theSalmonellapathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response todamlevels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed betweenSalmonellaserovars that are closely related at the DNA level.
First Complete Genome Sequence of TwoStaphylococcus epidermidisBacteriophages
Authors: Daniel Anu , Bonnen Penelope E. , Fischetti Vincent A.
Published: December 2006

Staphylococcus epidermidisis an important opportunistic pathogen causing nosocomial infections and is often associated with infections in patients with implanted prosthetic devices. A number of virulence determinants have been identified inS. epidermidis, which are typically acquired through horizontal gene transfer. Due to the high recombination potential, bacteriophages play an important role in these transfer events. Knowledge of phage genome sequences provides insights into phage-host biology and evolution. We present the complete genome sequence and a molecular characterization of twoS. epidermidisphages, φPH15 (PH15) and φCNPH82 (CNPH82). Both phages belonged to theSiphoviridaefamily and produced stable lysogens. The PH15 and CNPH82 genomes displayed high sequence homology; however, our analyses also revealed important functional differences. The PH15 genome contained two introns, and in vivo splicing of phage mRNAs was demonstrated for both introns. Secondary structures for both introns were also predicted and showed high similarity to those ofStreptococcus thermophilusphage 2972 introns. An additional finding was differential superinfection inhibition between the two phages that corresponded with differences in nucleotide sequence and overall gene content within the lysogeny module. We conducted phylogenetic analyses on all knownSiphoviridae, which showed PH15 and CNPH82 clustering withStaphylococcus aureus, creating a novel clade within theS. aureusgroup and providing a higher overall resolution of the siphophage branch of the phage proteomic tree than previous studies. Until now, noS. epidermidisphage genome sequences have been reported in the literature, and thus this study represents the first complete genomic and molecular description of twoS. epidermidisphages.
Reversing the slow death of the clinical necropsy: developing the post of the Pathology Liaison Nurse
Authors: Limacher Eileen , Carr Urszula , Bowker Lesley , Ball Richard Y
Published: December 2006

The adult clinical necropsy has been declining for many years and is nearing extinction in many hospitals. In Norwich, to prevent this from occurring, a Pathology Liaison Nurse (PLN) was appointed, resulting in a modest reversal of the trend. In 2005, the number of adult clinical necropsies increased to 58 (clinical necropsy rate = 2.4%) from its nadir of 34 (clinical necropsy rate = 1.4%) in 2003. Moreover, consent is now much more likely to be full and to allow histopathological and other studies. The PLN ensures that consent is properly and fully obtained, in line with current legislation. She also plays an important role in arranging for feedback to be given by clinicians to the families after the examination, and in teaching and training Trust staff about death, bereavement, and related matters. This paper describes how the role of PLN was established and evaluated, and gives details of the current state of the adult clinical necropsy in Norwich.
The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NO• response of isolated rat aorta
Authors: Rietjens S. J. , Bast A. , de Vente J. , Haenen G. R. M. M.
Published: December 2006

The Mediterranean diet, which is abundant in antioxidants, is associated with a relatively low incidence of coronary heart disease. Olive oil and olives, which contain the antioxidants hydroxytyrosol, oleuropein, and tyrosol, are important components of this diet. In this study, the effects of oxidative stress on the nitric oxide radical (NO•)-mediated relaxation of rat aorta and the protection by these antioxidants were determined. Cumene hydroperoxide (CHP) was used to mimic oxidative stress induced by lipid hydroperoxides, which is mediated by the formation of hydroxyl radicals (OH•). CHP (300 μM) impaired the NO•-mediated relaxation of rat aorta by the acetylcholine receptor agonist carbachol ( P < 0.05). This was due to a reduction in NO• production. A diminished NO•-mediated relaxation disturbs the vascular tone and leads to a rise in blood pressure, which is a well-established risk factor for coronary heart disease. Hydroxytyrosol (10 μM) efficiently protected the aorta against the CHP-induced impairment of the NO•-mediated relaxation ( P < 0.05). Oleuropein, tyrosol, and homovanillic alcohol, a major metabolite of hydroxytyrosol, did not show protection. Moreover, hydroxytyrosol was found to be a potent OH• scavenger, which can be attributed to its catechol moiety. Because of its amphiphilic characteristics (octanol-water partitioning coefficient = 1.1), hydroxytyrosol will readily cross membranes and provide protection in the cytosol and membranes, including the water-lipid interface. The present study provides a molecular basis for the contribution of hydroxytyrosol to the benefits of the Mediterranean diet.
Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy
Authors: Barrick Cordelia J. , Rojas Mauricio , Schoonhoven Robert , Smyth Susan S. , Threadgill David W.
Published: December 2006

Left ventricular hypertrophy (LVH), a risk factor for cardiovascular morbidity and mortality, is commonly caused by essential hypertension. Three geometric patterns of LVH can be induced by hypertension: concentric remodeling, concentric hypertrophy, and eccentric hypertrophy. Clinical studies suggest that different underlying etiologies, genetic modifiers, and risk of mortality are associated with LVH geometric patterns. Since pressure overload-induced LVH can be modeled experimentally using transverse aortic constriction (TAC) and since C57BL/6J (B6) and 129S1/SvImJ (129S1) strains, which have different baseline cardiovascular phenotypes, are commonly used, we conducted serial echocardiographic studies to assess cardiac function up to 8 wk of post-TAC in male B6, 129S1, and B6129F1 (F1) mice. B6 mice had an earlier onset and more pronounced impairment in contractile function, with corresponding left and right ventricular dilatation, fibrosis, change in expression of hypertrophy marker, and increased liver weights at 5 wk of post-TAC. These observations suggest that B6 mice had eccentric hypertrophy with systolic dysfunction and right-sided heart failure. In contrast, we found that 129S1 and F1 mice delayed transition to decompensated heart failure, with 129S1 mice exhibiting preserved systolic function until 8 wk of post-TAC and relatively mild alterations in histology and markers of hypertrophy at 5 wk post-TAC. Consistent with concentric hypertrophy, our results show that these strains manifest different cardiac responses to pressure overload in a time-dependent manner and that genetic susceptibility to initial concentric hypertrophy is dominant to eccentric hypertrophy. These results also imply that genetic background differences can complicate interpretation of TAC studies when using mixed genetic backgrounds.
Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts
Authors: Makazan Zhanna , Saini Harjot K. , Dhalla Naranjan S.
Published: December 2006

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [ N-acetyl-l-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H2O2) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.
Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling
Authors: Shao Zuo-Hui , Chang Wei-Tien , Chan Kim Chai , Wojcik Kim R. , Hsu Chin-Wang , Li Chang-Qing , Li Juan , Anderson Travis , Qin Yimin , Becker Lance B. , Hamann Kimm J. , Vanden Hoek Terry L.
Published: December 2006

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 ± 3.4% cell death. Hypothermia induction to 25°C was most protective (14.3 ± 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 ± 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 ± 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor Nω-nitro-l-arginine methyl ester (200 μM) reversed these changes and abrogated hypothermia protection. In addition, the PKCε inhibitor myr-PKCε v1-2 (5 μM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cε; nitric oxide synthase.
Cav3.1 (α1G) controls von Willebrand factor secretion in rat pulmonary microvascular endothelial cells
Authors: Zhou Chun , Chen Hairu , Lu Fengmin , Sellak Hassan , Daigle Jonathan A. , Alexeyev Mikhail F. , Xi Yaguang , Ju Jingfang , van Mourik Jan A. , Wu Songwei
Published: December 2006

The T-type Ca2+channel Cav3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Cav3.1 in thrombin-induced Weibel-Palade body exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a green fluorescent protein (GFP)-tagged VWF chimera, we examined the real-time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. Whereas thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in the number of VWF-GFP-containing vesicles was nearly abolished by the T-type Ca2+channel blocker mibefradil as well as by Cav3.1 gene silencing with small hairpin RNA. Expression of recombinant Cav3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca2+entry, which is sensitive to mibefradil. Together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca2+or cAMP, and support the hypothesis that activation of Cav3.1 T-type Ca2+channels in PMVECs provides a unique cytosolic Ca2+source important for Gq-linked agonist-induced VWF release.
Levinas and Adorno: Universalizing the Jew after Auschwitz
Authors: Eisenstadt Oona
Published: December 2006

The paper uses a comparison of Levinas and Adorno to raise certain questions about the former's thought. Both thinkers utilize Jewish experience as a trope for what eludes systematic conceptualization (particularly the conceptualization of history): the universalization and varying degrees of abstraction required to make this move form the content of the paper's central analysis. However, this analysis is framed in a wider argument about the way in which Levinas is an innovative thinker, the problems this innovation raises for the question of the eternal relevance of his ideas, and the influence a unique aspect of his thought has had on contemporary philosophers who seek to update him.
Verdict and Sentence: Cover and Levinas on the Robe of Justice
Authors: Gibbs Robert
Published: December 2006

Few problems are as challenging to Levinas's ethics as the tension or even chiasm that opens between the ethics in relation to the face and the claims of the third. This paper offers a reading of the role of the judge in court as the model for understanding the relation of these two aspects of justice. I make reference to an essay by the legal theorist Robert Cover that explored the violence of the courtroom. He shows how society contains appropriate violence by framing the institutions of the court. Levinas then appears in his repeated citation of a Talmudic text about the judge not facing the defendant in a court. Through a careful reading of the Talmudic text and the Biblical texts upon which it draws, we can see Levinas sorting out the different kinds of responsibility to which the judge responds. The paper ends with a brief reflection on how engaging with the specificity of a text offers new methodological resources for philosophical reflection.