bluuebunny/crossref_metadata_2025 huggingface dataset:
rpoS
Mutations and Loss of General Stress Resistance in
Escherichia coli
Populations as a Consequence of Conflict between Competing Stress Responses
Authors: Notley-McRobb Lucinda , King Thea , Ferenci Thomas
Published: April 2009

The general stress resistance of
Escherichia coli
is controlled by the RpoS sigma factor (φ
S
), but mutations in
rpoS
are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing
rpoS
was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by
rpoS
mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of
rpoS
null mutations and loss of general stress resistance. The rate of takeover by
rpoS
mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher φ
S
levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on φ
70
and involved in the hunger response to nutrient limitation. Indeed, the mutation of
rpoS
led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence,
rpoS
polymorphism in
E. coli
populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than φ
S
for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of
rpoS
mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of
rpoS
mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.
Cytochrome
aa
3
in
Haloferax volcanii
Authors: Tanaka Mikiei , Ogawa Naohide , Ihara Kunio , Sugiyama Yasuo , Mukohata Yasuo
Published: April 2009

A cytochrome in an extremely halophilic archaeon,
Haloferax volcanii
, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of
a
-type cytochromes and a CN
−
complex spectrum that indicates the presence of heme
a
and heme
a
3
. This cytochrome
aa
3
consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome
c
partially purified from the bacterial membrane fraction was oxidized by the cytochrome
aa
3
, providing physiological evidence for electron transfer from cytochrome
c
to cytochrome
aa
3
in archaea.
Molecular and Physical Characterization of
Burkholderia mallei
O Antigens
Authors: Burtnick Mary N. , Brett Paul J. , Woods Donald E.
Published: April 2009

Burkholderia mallei
lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against
B. pseudomallei
LPS; however, we observed that
B. mallei
LPS does not react with a monoclonal antibody (Pp-PS-W) specific for
B. pseudomallei
O polysaccharide (O-PS). In this study, we identified the O-PS biosynthetic gene cluster from
B. mallei
ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism.
Rex-Centric Mutualism
Authors: Slavcev Roderick A. , Hayes Sidney
Published: April 2009

We asked whether Rex exclusion encoded by a lambda prophage confers a protective or a cell-killing phenotype. We found that the Rex system can channel lysogenic cells into an arrested growth phase that gives an overall protective ability to the host despite some associated killing.
NADPH-Dependent
l
-Sorbose Reductase Is Responsible for
l
-Sorbose Assimilation in
Gluconobacter suboxydans
IFO 3291
Authors: Shinjoh Masako , Tazoe Masaaki , Hoshino Tatsuo
Published: April 2009

The NADPH-dependent
l
-sorbose reductase (SR) of
l
-sorbose-producing
Gluconobacter suboxydans
IFO 3291 contributes to intracellular
l
-sorbose assimilation. The gene disruptant showed no SR activity and did not assimilate the once-produced
l
-sorbose, indicating that the SR functions mainly as an
l
-sorbose-reducing enzyme in vivo and not as a
d
-sorbitol-oxidizing enzyme.
ZupT Is a Zn(II) Uptake System in
Escherichia coli
Authors: Grass Gregor , Wong Marco D. , Rosen Barry P. , Smith Ron L. , Rensing Christopher
Published: April 2009

Escherichia coli zupT
(
ygiE
), encoding a ZIP family member, mediated zinc uptake. Growth of cells disrupted in both
zupT
and the
znuABC
operon was inhibited by EDTA at a much lower concentration than a single mutant or the wild type. Cells expressing ZupT from a plasmid exhibited increased uptake of
65
Zn
2+
.
The Flavoprotein MrsD Catalyzes the Oxidative Decarboxylation Reaction Involved in Formation of the Peptidoglycan Biosynthesis Inhibitor Mersacidin
Authors: Majer Florian , Schmid Dietmar G. , Altena Karsten , Bierbaum Gabriele , Kupke Thomas
Published: April 2009

The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II. Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA. We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue. MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family). Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD. In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein. HFCD enzymes are characterized by a conserved His residue which is part of the active site. Exchange of this His residue for Asn led to inactivation of MrsD. The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities.
PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B
12
-Dependent Degradation of 1,2-Propanediol in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Havemann Gregory D. , Sampson Edith M. , Bobik Thomas A.
Published: April 2009

Salmonella enterica
forms polyhedral organelles involved in coenzyme B
12
-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B
12
-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (
pdu
) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the
pduA
gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B
12
-dependent 1,2-propanediol degradation. A His
6
-PduA fusion protein was purified from a recombinant
Escherichia coli
strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar
pduA
mutations were unable to form organelles. These results show that the
pduA
gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar
pduA
mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar
pduA
deletion mutant grew faster than the wild-type strain at low vitamin B
12
concentrations. These results suggest that the polyhedral organelles formed by
S. enterica
during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B
12
, but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.
Reconstitution of the Trimethylamine Oxide Reductase Regulatory Elements of
Shewanella oneidensis
in
Escherichia coli
Authors: Gon Stéphanie , Patte Jean-Claude , Dos Santos Jean-Philippe , Méjean Vincent
Published: April 2009

Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors. In
Shewanella
species, the TMAO reductase respiratory system is encoded by the
torECAD
operon. We showed that production of the TMAO reductase of
S. oneidensis
was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (
torSTR
) encoding a complex two-component regulatory system was present downstream of the
torECAD
operon. We introduced the
torSTR
gene cluster into
Escherichia coli
and showed that this regulatory gene cluster is involved in TMAO induction of the
torE
promoter but plays no role in the oxygen control. The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the
tor
structural operon. By deletion analysis, we confirmed that the TorR operator site is required for induction of the
tor
structural promoter. As the TMAO regulatory system of
S. oneidensis
is homologous to that of
E. coli
, we investigated a possible complementation between the TMAO regulatory components of the two bacteria. Interestingly, TorS
ec
, the TMAO sensor of
E. coli
, was able to transphosphorylate TorR
so
, the TMAO response regulator of
S. oneidensis
.
Salmonella enterica
Serovar Typhimurium Resistance to Bile: Identification and Characterization of the
tolQRA
Cluster
Authors: Prouty Angela M. , Van Velkinburgh Jennifer C. , Gunn John S.
Published: April 2009

Salmonella enterica
serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a
phoP
mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative
Salmonella orf1-tolQRA
operon, with insertions in
tolQ
,
orf1
, and a gene upstream of the
orf1-tolQRA
operon not previously associated with Tol function (
orfX
). Through the use of transcriptional fusions, none of the putative
tol
(or
tol
-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (
orfX
,
orf1
,
tolQRA
) are predicted to be cotranscribed. This is the first identification of
Salmonella
serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these
tol
genes demonstrate significant differences from their
Escherichia coli
homologs.
Allelic Diversity and Population Structure in
Vibrio cholerae
O139 Bengal Based on Nucleotide Sequence Analysis
Authors: Farfán M. , Miñana-Galbis D. , Fusté M. C. , Lorén J. G.
Published: April 2009

Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of
Vibrio cholerae
, has been carried out for a collection of 29
V. cholerae
O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same
V. cholerae
O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the
V. cholerae
O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.
Trinucleotide GAA Repeats Dictate pMGA Gene Expression in
Mycoplasma gallisepticum
by Affecting Spacing between Flanking Regions
Authors: Liu Li , Panangala Victor S. , Dybvig Kevin
Published: April 2009

The pMGA genes of the avian respiratory pathogen
Mycoplasma gallisepticum
encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless
lacZ
gene and transformed into
M. gallisepticum
by using transposon Tn
4001
as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal) to monitor β-galactosidase activity. For the transformants of
M. gallisepticum
containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers,
lacZ
was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.
Positive Growth Rate-Dependent Regulation of the
pdxA
,
ksgA
, and
pdxB
Genes of
Escherichia coli
K-12
Authors: Pease Andrew J. , Roa Benjamin R. , Luo Wen , Winkler Malcolm E.
Published: April 2009

We found that transcription of the
pdxA
and
pdxB
genes, which mediate steps in the biosynthesis of the essential coenzyme pyridoxal 5"-phosphate, and the
ksgA
gene, which encodes an rRNA modification enzyme and is partly cotranscribed with
pdxA
, is subject to positive growth rate regulation in
Escherichia coli
K-12. The amounts of the
pdxA-ksgA
cotranscript and
pdxB-
and
ksgA-
specific transcripts and expression from
pdxA-
and
pdxB
-
lacZ
fusions increased as the growth rate increased. The half-lives of
ksgA-
and
pdxB-
specific transcripts were not affected by the growth rate, whereas the half-life of the
pdxA-ksgA
cotranscript was too short to be measured accurately. A method of normalization was applied to determine the amount of mRNA synthesized per gene and the rate of protein accumulation per gene. Normalization removed an apparent anomaly at fast growth rates and demonstrated that positive regulation of
pdxB
occurs at the level of transcription initiation over the whole range of growth rates tested. RNA polymerase limitation and autoregulation could not account for the positive growth rate regulation of
pdxA
,
pdxB
, and
ksgA
transcription. On the other hand, growth rate regulation of the amount of the
pdxA-ksgA
cotranscript was abolished by a
fis
mutation, suggesting a role for the Fis protein. In contrast, the
fis
mutation had no effect on
pdxB-
or
ksgA
-specific transcript amounts. The amounts of the
pdxA-ksgA
cotranscript and
ksgA-
specific transcript were repressed in the presence of high intracellular concentrations of guanosine tetraphosphate; however, this effect was independent of
relA
function for the
pdxA-ksgA
cotranscript. Amounts of the
pdxB
-specific transcript remained unchanged during amino acid starvation in wild-type and
relA
mutant strains.
NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes
Authors: Yu Jian-Lin , Grinius Leo , Hooper David C.
Published: April 2009

Overexpression of NorA, an endogenous efflux transporter ofStaphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. ThenorAgene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed inEscherichia colicells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K+-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.
Effects of a Squalene Epoxidase Inhibitor, Terbinafine, on Ether Lipid Biosyntheses in a Thermoacidophilic Archaeon,
Thermoplasma acidophilum
Authors: Kon Takahide , Nemoto Naoki , Oshima Tairo , Yamagishi Akihiko
Published: April 2009

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon
Thermoplasma acidophilum
. Terbinafine was found to inhibit the growth of
T. acidophilum
in a concentration-dependent manner. When growing
T. acidophilum
cells were pulse-labeled with [2-
14
C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in
T. acidophilum
.
Quantitative Assessment of Oxygen Availability: Perceived Aerobiosis and Its Effect on Flux Distribution in the Respiratory Chain of
Escherichia coli
Authors: Alexeeva Svetlana , Hellingwerf Klaas J. , Teixeira de Mattos M. Joost
Published: April 2009

Despite a large number of studies on the role of oxygen in cellular processes, there is no consensus as to how oxygen availability to the cell should be defined, let alone how it should be quantified. Here, a quantitative definition for oxygen availability (perceived aerobiosis) is presented; the definition is based on a calibration with reference to the minimal oxygen supply rate needed for fully oxidative catabolism (i.e., complete conversion of the energy source to CO
2
and water for glucose-limited conditions). This quantitative method is used to show how steady-state electron fluxes through the alternative cytochrome oxidases of
Escherichia coli
are distributed as a function of the extent of aerobiosis of glucose-limited chemostat cultures. At low oxygen availability the electron flux is mainly via the high-affinity cytochrome
bd
oxidase, and, at higher oxygen availability, a similar phenomenon occurs but now via the low-affinity cytochrome
bo
oxidase. The main finding is that the catabolic activities of
E. coli
(and specifically its respiratory activity) are affected by the actual oxygen availability per unit of biomass rather than by the residual dissolved oxygen concentration of the culture.
Posttranscriptional Activation of the Transcriptional Activator Rob by Dipyridyl inEscherichia coli
Authors: Rosner Judah L. , Dangi Bindi , Gronenborn Angela M. , Martin Robert G.
Published: April 2009

The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present perEscherichia colicell, they activate promoters of therobregulon poorly. We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of variousrobregulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on therobpromoter was observed. However, dipyridyl enhanced Rob's activity even whenrobwas regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.
Directed Mutagenesis and Plasmid-Based Complementation in the Methanogenic Archaeon
Methanosarcina acetivorans
C2A Demonstrated by Genetic Analysis of Proline Biosynthesis
Authors: Zhang Jun Kai , White Andrea K. , Kuettner H. Craig , Boccazzi Paolo , Metcalf William W.
Published: April 2009

We report here the first use of directed mutagenesis in
Methanosarcina acetivorans
C2A. The method employs homologous recombination-mediated gene replacement and was used to construct a variety of proline auxotrophs with mutations in the
proABC
locus. Each mutation was also complemented in
trans
with autonomously replicating
Methanosarcina
-
Escherichia
plasmid shuttle vectors.
Bypass of A- and B-Signaling Requirements for
Myxococcus xanthus
Development by Mutations in
spdR
Authors: Tse Hubert , Gill Ronald E.
Published: April 2009

Mutations in
spdR
, previously reported to bypass the developmental requirement for B-signaling in
Myxococcus xanthu
s, also bypass the requirement for A-signaling but not C-, D-, or E-signaling. Mutations in
spdR
restored nearly wild-type levels of sporulation to representative A-signal-deficient mutants carrying
asgA476, asgB480
, and
asgC767
and improved the quality of fruiting body formation in the
asgB480
mutant. The defect in A-factor production by the
asgB480
mutant was not restored in the
spdR2134 asgB480
double mutant.
Mapping of
Myxococcus xanthus
Social Motility
dsp
Mutations to the
dif
Genes
Authors: Lancero Hope , Brofft Jennifer E. , Downard John , Birren Bruce W. , Nusbaum Chad , Naylor Jerome , Shi Wenyuan , Shimkets Lawrence J.
Published: April 2009

Myxococcus xanthus dsp
and
dif
mutants have similar phenotypes in that they are deficient in social motility and fruiting body development. We compared the two loci by genetic mapping, complementation with a cosmid clone, DNA sequencing, and gene disruption and found that 16 of the 18
dsp
alleles map to the
dif
genes. Another
dsp
allele contains a mutation in the
sglK
gene. About 36.6 kb around the
dsp-dif
locus was sequenced and annotated, and 50% of the genes are novel.
Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ
1
H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from
cis
-Dienelactone
Authors: Pieper Dietmar H. , Pollmann Katrin , Nikodem Patricia , Gonzalez Bernardo , Wray Victor
Published: April 2009

A
1
H nuclear magnetic resonance (
1
H NMR) assay was used to study the enzymatic transformation of
cis
-dienelactone, a central intermediate in the degradation of chloroaromatics. It was shown that the product of the
cis
-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate. Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2-en-4-olide. Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into
cis
-acetylacrylate. One-dimensional
1
H NMR in
1
H
2
O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products.
Novel Group I Intron in the tRNA
Leu
(UAA) Gene of a γ-Proteobacterium Isolated from a Deep Subsurface Environment
Authors: Vepritskiy Alexey A. , Vitol Inna A. , Nierzwicki-Bauer Sandra A.
Published: April 2009

A group I intron has been found to interrupt the anticodon loop of the tRNA
Leu
(UAA) gene in a bacterium belonging to the γ-subdivision of
Proteobacteria
and isolated from a deep subsurface environment. The subsurface isolate SMCC D0715 was identified as belonging to the genus
Pseudomonas
. The group I intron from this isolate is the first to be reported for γ-proteobacteria, and the first instance of a tRNA
Leu
(UAA) group I intron to be found in a group of bacteria other than cyanobacteria. The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10. The D0715 intron possesses the 11-nt motif CCUACG … UAUGG in its P8 region, a feature not common in bacterial introns. To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements.
Endocarditis Due to
Neisseria bacilliformis
in a Patient with a Bicuspid Aortic Valve
Authors: Masliah-Planchon Julien , Breton Guillaume , Jarlier Vincent , Simon Anne , Benveniste Olivier , Herson Serge , Drieux Laurence
Published: April 2009

We report a case of endocarditis due to the rod-shaped
Neisseria
species
Neisseria bacilliformis
. The phenotypic characterization of this recently characterized bacteria is difficult, and the identification requires the sequencing of the 16S rRNA gene. The resolution of the disease was complete after appropriate antibiotic therapy, and surgery was not required.
Evaluation of Impact of Serial Hepatitis B Virus DNA Levels on Development of Hepatocellular Carcinoma
Authors: Chan Henry Lik-Yuen , Wong Vincent Wai-Sun , Wong Grace Lai-Hung , Chim Angel Mei-Ling , Lai Larry Hin , Sung Joseph Jao-Yiu
Published: April 2009

We aimed to investigate the impact of hepatitis B virus (HBV) DNA on the development of hepatocellular carcinoma (HCC). We conducted a case/control study based on 506 chronic HBV patients followed up since 1997. Forty-one patients developed HCC, and each of them was age and gender matched with two simultaneously recruited controls without HCC. HBV DNA was measured at the initial visit, at yearly intervals, and at the last visit. Patient age at the time of HCC development was 55 ± 9 years. Forty-nine (40%) patients experienced antiviral treatment. The median time from diagnosis to the development of HCC was 17 months, and the control patients were followed for 92 months. At the trough level (defined as lowest level among all studied visits), more (27 patients; 66%) HCC patients had HBV DNA levels of >10,000 copies/ml than the controls (17 patients; 21%). The area under the receiver operating characteristic curve of the trough log HBV DNA level for HCC was 0.79 (95% confidence interval [CI], 0.69 to 0.89). Trough log HBV DNA (odds ratio, 11.4; 95% CI, 3.6 to 37.6;
P
< 0.0001) and liver cirrhosis (odds ratio, 11.4; 95% CI, 3.6 to 36.2;
P
< 0.0001) levels were independently associated with HCC after an adjustment for age, gender, antiviral treatment, and HBV genotype. The difference in the trough HBV DNA level was more obvious among untreated patients (5.7 ± 1.4 log copies/ml in HCC patients versus 3.2 ± 1.3 log copies/ml in control patients;
P
< 0.0001) than among those who had received antiviral treatment (3.0 ± 1.4 log copies/ml in HCC patients versus 2.5 ± 0.9 log copies/ml in control patients;
P
= 0.38). A high trough HBV DNA level was associated with a higher risk of HCC. Whether antiviral treatment could prevent HCC was uncertain.
Feasibility of the GenoType MTBDR
sl
Assay for Fluoroquinolone, Amikacin-Capreomycin, and Ethambutol Resistance Testing of
Mycobacterium tuberculosis
Strains and Clinical Specimens
Authors: Hillemann Doris , Rüsch-Gerdes Sabine , Richter Elvira
Published: April 2009

The new GenoType
Mycobacterium tuberculosis
drug resistance second line (MTBDR
sl
) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in
Mycobacterium tuberculosis
strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDR
sl
assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDR
sl
assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDR
sl
assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.
Outbreak of Nontuberculous Mycobacterial Subcutaneous Infections Related to Multiple Mesotherapy Injections
Authors: Carbonne Anne , Brossier Florence , Arnaud Isabelle , Bougmiza Iheb , Caumes Eric , Meningaud Jean-Paul , Dubrou Sylvie , Jarlier Vincent , Cambau Emmanuelle , Astagneau Pascal
Published: April 2009

We describe an outbreak of severe subcutaneous infections due to nontuberculous mycobacteria following mesotherapy. Epidemiological studies and molecular comparisons of
Mycobacterium chelonae
strains from different patients and the environment suggested that contamination may be associated with inappropriate cleaning of the multiple-injection device with tap water.
PediatricHelicobacter pyloriIsolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles
Authors: Talarico Sarah , Gold Benjamin D. , Fero Jutta , Thompson Dexter T. , Guarner Jeannette , Czinn Steven , Salama Nina R.
Published: April 2009

Helicobacter pyloristrains display remarkable genetic diversity, and the presence of strains bearing the toxigenicvacAs1 allele, a completecagpathogenicity island (PAI),cagAalleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterizedH. pyloristrains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multipleH. pyloriclones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. ThecagAandvacAalleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for thecagAgene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a completecagPAI. Thirty patients (64%) were infected with a strain having thevacAs1 allele, and 28 patients (60%) had thebabAgene. The presence of a functionalcagPAI was correlated with ulcer disease (P= 0.0095). In spite of declining rates ofH. pyloriinfection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected withH. pyloristrains lacking thecagPAI, but the presence of a completecagPAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.
Spontaneous Bacterial Pericarditis with Tamponade Due to
Ureaplasma
spp
Authors: Tarrant William P. , Gonzalez-Berjon Jose M. , Cernoch Patricia L. , Olsen Randall J. , Musser James M.
Published: April 2009

Infectious pericardial effusion with tamponade is an uncommon but life-threatening disease. We report an unusual case of spontaneous
Ureaplasma
pericardial effusion with tamponade associated with pneumonia, pleural effusion, and urinary tract infection. All published cases of clinically invasive
Ureaplasma
infections in the adult population are also reviewed.
High Diversity ofMycobacterium tuberculosisGenotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates
Authors: Stavrum Ruth , Mphahlele Matsie , Øvreås Kristi , Muthivhi Tshilidzi , Fourie P. Bernard , Weyer Karin , Grewal Harleen M. S.
Published: April 2009

The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixedMycobacterium tuberculosisinfections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution ofM.tuberculosisgenotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixedM.tuberculosissubpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10;P= <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixedM.tuberculosissubpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.
Complete Genome Sequence and Phylogenetic Relatedness of Hepatitis B Virus Isolates in Papua, Indonesia
Authors: Utsumi Takako , Lusida Maria Inge , Yano Yoshihiko , Nugrahaputra Victor Eka , Amin Mochamad , Juniastuti  , Soetjipto  , Hayashi Yoshitake , Hotta Hak
Published: April 2009

Each hepatitis B virus (HBV) genotype and subgenotype is associated with a particular geographic distribution, ethnicity, and anthropological history. Our previous study showed the novel HBV subgenotypes C6 (HBV/C6) and D6 (HBV/D6), based on the S gene sequences of isolates in Papua, Indonesia. The present study investigated the complete genome sequence of 22 strains from Papua and subjected them to molecular evolutionary analysis. A phylogenetic analysis revealed that 9 out of 22 strains were classified as HBV/C6, 3 strains as HBV/D6, and 9 strains as HBV/B3. A particular strain positioned between HBV/B3 and HBV/B5 remained unclassifiable into any known subgenotypes. This strain showed high homology with HBV/C5 from the Philippines in the core region and was thought to have undergone genetic recombination with HBV/C5. Further studies are needed to determine whether this strain belongs to a new subgenotype of HBV/B. Based on the amino acid alignment, HBV/C6 has subgenotype specific variations (G18V and V47M) in the S region. HBV/C6 strains were more closely related in terms of evolutionary distance to strains from the east Asia and Pacific regions than those found in southeast Asia. HBV/D6 strains were most closely related to strains from the Western countries (HBV/D3) rather than those from Asia and Papua New Guinea. In conclusion, we have confirmed by complete sequence analysis that two novel HBV subgenotypes, HBV/C6 and HBV/D6, are prevalent in Papua, Indonesia.
Activity of Contemporary Antifungal Agents, Including the Novel Echinocandin Anidulafungin, Tested against
Candida
spp.,
Cryptococcus
spp., and
Aspergillus
spp.: Report from the SENTRY Antimicrobial Surveillance Program (2006 to 2007)
Authors: Messer Shawn A. , Moet Gary J. , Kirby Jeffrey T. , Jones Ronald N.
Published: April 2009

Results from the SENTRY international fungal surveillance program for 2006 to 2007 are presented. A total of 1,448
Candida
sp., 49
Aspergillus fumigatus
, and 33
Cryptococcus neoformans
isolates were obtained from infected sterile-site sources in patients on five continents. Reference susceptibility was determined for anidulafungin, caspofungin, 5-flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, and amphotericin B by CLSI methods.
Escherichia coli
ehl1
Gene-Positive Serotype O18ac:H31 Associated with an Outbreak of Diarrhea in a Neonatal Nursery in Neuquén City, Argentina
Authors: Chinen Isabel , Rivas Marta , Soriano Viviana , Miliwebsky Elizabeth , Galvez Gabriel Fernández , Chillemi Germán , Baschkier Ariela , Wang Gehua , Caldeira Richard , Woodward David L. , Rodgers Frank G.
Published: April 2009

Between 9 October and 12 November 1996, an outbreak of bloody diarrhea occurred in the neonatal nursery ward of the Policlínico Neuquén, in Neuquén, a city in the southwestern region of Argentina. Seven patients of the intermediate care unit were affected. Isolates of
Escherichia coli
O18ac:H31 that were non-lactose and -sorbitol fermenting were recovered from outbreak cases. Although the strains were negative for a number of virulence factors typically found in diarrheagenic groups of
E. coli
, all isolates from the present neonatal outbreak possessed the enterohemolysin gene,
ehl1
. All isolates showed resistance to the antibiotics ampicillin and chloramphenicol. These isolates showed a low adherence property in HeLa cells without any recognizable pattern. In order to evaluate the outbreak dissemination in the neonatology ward, a prevalence study was conducted on 13 November. Stool specimens were obtained from 16 neonates hospitalized in the sector and from 33 medical staff members.
E. coli
isolates with identical biochemical characteristics of the outbreak strain were recovered from 11 of 16 inpatients and from 4 of 33 staff members during the prevalence study. A total of 15
E. coli
strains recovered both from the outbreak and the prevalence study were processed by random amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE). By RAPD-PCR 14 of 15 strains showed patterns with 85 to 100% similarity, and by PFGE these strains were identical, each showing a unique pattern with 15 bands between 40 and 400 kb. One strain isolated from a nurse during the prevalence study presented a pattern not related to the others, and this was characterized as
E. coli
O81:HNM resistant to ampicillin only and negative for all the virulence factors studied. This outbreak occurred despite strict regulations in place to prevent cross-infection in the hospital. Postoutbreak prevalence studies were performed weekly thereafter without detecting new cases.
Posttrabeculectomy Endophthalmitis Caused by
Moraxella nonliquefaciens
Authors: Laukeland Helene , Bergh Kåre , Bevanger Lars
Published: April 2009

Moraxella nonliquefaciens
, a commensal organism of the upper respiratory tract, is generally considered to have low pathogenic potential. We report here two cases of severe endophthalmitis occurring 9 years and 2 months after glaucoma filtration surgery, respectively. Apart from sulfonamide, very low MICs were recorded for several antibiotics tested. Identification was based on phenotypic characteristics in combination with sequencing of the 16S rRNA gene.
Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments
Authors: Kudoh Ayumi , Iwahori Satoko , Sato Yoshitaka , Nakayama Sanae , Isomura Hiroki , Murata Takayuki , Tsurumi Tatsuya
Published: April 2009

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.
The Crystal Structures of Chikungunya and Venezuelan Equine Encephalitis Virus nsP3 Macro Domains Define a Conserved Adenosine Binding Pocket
Authors: Malet Hélène , Coutard Bruno , Jamal Saïd , Dutartre Hélène , Papageorgiou Nicolas , Neuvonen Maarit , Ahola Tero , Forrester Naomi , Gould Ernest A. , Lafitte Daniel , Ferron Francois , Lescar Julien , Gorbalenya Alexander E. , de Lamballerie Xavier , Canard Bruno
Published: April 2009

Macro domains (also called “X domains”) constitute a protein module family present in all kingdoms of life, including viruses of theCoronaviridaeandTogaviridaefamilies. Crystal structures of the macro domain from the Chikungunya virus (an “Old World” alphavirus) and the Venezuelan equine encephalitis virus (a “New World” alphavirus) were determined at resolutions of 1.65 and 2.30 Å, respectively. These domains are active as adenosine di-phosphoribose 1″-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1″-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.