bluuebunny/crossref_metadata_2025 huggingface dataset:
Species-Specific Monoclonal Antibodies for Rapid Identification ofBartonella quintana
Authors: Liang Zhongxing , Raoult Didier
Published: April 2009

Seven species-specific monoclonal antibodies (MAbs) toBartonella quintanawere produced and characterized. The MAbs were of the immunoglobulin G class and reacted only with 13B. quintanastrains in indirect microimmunofluorescence and Western immunoblotting assays. They did not react with eight otherBartonellaspp., includingBartonella henselae, the most closely related species, and a selected MAb did also not react with nine other strains of gram-negative bacteria. The MAbs reacted mainly with a 34-kDa protein epitope ofB. quintanawhich was shown to be species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four of five body lice experimentally infected withB. quintanawere found to be positive for the organism in microimmunofluorescence assays with one MAb. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification ofB. quintanaand the diagnosis of infections due to the microorganism.
Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
Authors: Zhang Zhu-Xu , Lazdina Una , Chen Margaret , Peterson Darrell L. , Sällberg Matti
Published: April 2009

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.
Measurement of Antinuclear Antibodies: Assessment of Different Test Systems
Authors: Kern P. , Kron M. , Hiesche K.
Published: April 2009

The performance of rat liver and HEp-2 in the detection of antinuclear antibodies (ANA) was studied by two independent sites and compared against an ANA enzyme immunoassay (EIA) screen and EIA systems for the measurement of antibodies to double-stranded DNA (dsDNA) and ENA. Sixty-two sera from patients with connective tissue disease (CTD) and 398 from controls suffering from other disorders were included. The level of agreement was, for HEp-2 and rat liver (within one site), 82.0% (ANA positive/ANA negative) and 51.0% (ANA pattern); and for HEp2- and HEp-2 (between sites), 71.8 and 86.5%. On sera with the ANA homogeneous pattern, the measurement of anti-ENA EIA added little to the detection rate with anti-dsDNA EIA alone. On ANA speckled sera, the EIA reactivity depended on the reaction of the mitotic cells: while sera with positive mitoses reacted similarly to ANA homogeneous sera, in those with negative mitoses the measurement of anti-ENA added about 10% to the detection rate achieved with anti-dsDNA alone. The measurement of anti-Scl-70 and anti-Jo-1 did not markedly improve the positive rate with classical ENA (anti-SSA, -SSB, -Sm, and -RNP) alone, raising doubts about the cost efficiency of including these measurements in unselected sera. The ANA EIA identified patients with CTD at a rate similar to that for rat liver and HEp-2. However, up to 98% of the sera found to be negative by ANA EIA but positive by use of rat liver and HEp-2 were from controls. Thus, the ANA EIA may possible be used as an alternative screen, particularly in laboratories with a high frequency of sera from patients not suffering from CTD.
Detection of
Francisella tularensis
in Biological Specimens Using a Capture Enzyme-Linked Immunosorbent Assay, an Immunochromatographic Handheld Assay, and a PCR
Authors: Grunow Roland , Splettstoesser Wolf , McDonald Sahra , Otterbein Christian , O'Brien Tom , Morgan Cecilia , Aldrich Jennifer , Hofer Erwin , Finke Ernst-Jürgen , Meyer Hermann
Published: April 2009

The early detection of
Francisella tularensis
, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of
Francisella tularensis
subsp.
holarctica
and
Francisella tularensis
subsp.
tularensis
. No cross-reactivity with
Francisella tularensis
subsp.
novicida
,
Francisella philomiragia
, and a panel of other possibly related bacteria, including
Brucella
spp.,
Yersinia
spp.,
Escherichia coli
, and
Burkholderia
spp., was observed. The detection limit of the assay was 10
3
to 10
4
bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of
F. tularensis
, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect
F. tularensis
in tissue samples of European brown hares (
Lepus europaeus
). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with
F. tularensis
, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.
Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis
Authors: Cacere Camila R. , Mendes-Giannini Maria J. S. , do Valle Antonio Carlos F. , Duarte Alberto J. S. , Benard Gil
Published: April 2009

To better understand the T-cell hyporesponsiveness of patients with paracoccidioidomycosis, we tested the hypothesis that the T cells were committed to apoptosis. We show here that T cells of patients with paracoccidioidomycosis overexpress caspase 9 and caspase 8 but express low Bcl-2 levels and that interleukin-2 was unable to revert the hyporesponsiveness. These data suggest that the T cells would in vivo be driven to a tolerant state and apoptosis.
Evaluation of a Commercial Blocking Enzyme-Linked Immunosorbent Assay To Detect Avian Influenza Virus Antibodies in Multiple Experimentally Infected Avian Species
Authors: Brown Justin D. , Stallknecht David E. , Berghaus Roy D. , Luttrell M. Page , Velek Katherine , Kistler Whitney , Costa Taiana , Yabsley Michael J. , Swayne David
Published: April 2009

Wild birds of the orders
Anseriformes
and
Charadriiformes
are the natural reservoirs for avian influenza (AI) viruses. Traditionally, AI virus surveillance in wild birds has relied on virus identification strategies, including virus isolation and detection. To evaluate the accuracy of a commercial blocking enzyme-linked immunosorbent assay (bELISA) and the agar gel immunodiffusion (AGID) test for detection of antibodies in wild birds, which is indicative of AI virus infection, we tested 281 serum samples from various wild avian species that were experimentally infected with AI viruses. Included in these samples were 178 samples from birds with confirmed AI virus infections (122 infected with low-pathogenic AI [LPAI] viruses and 56 infected with highly pathogenic AI [HPAI] viruses) and 103 samples from birds that were uninfected, negative controls. The sensitivities of the bELISA and the AGID test were 0.820 (95% confidence interval [95% CI], 0.756 to 0.874) and 0.674 (95% CI, 0.600 to 0.742), respectively. Both tests had an estimated specificity of 1.00 (95% CI, 0.965 to 1.00). The bELISA was significantly more sensitive than the AGID test for both LPAI virus- and HPAI virus-infected birds. Both assays, however, had a higher sensitivity for birds infected with HPAI virus than for birds infected with LPAI virus. These results demonstrate the potential utility of the bELISA for detection of antibodies to both LPAI and HPAI viruses in multiple avian species, representing five avian orders and 17 genera. Additional studies are warranted to further evaluate the utility of the bELISA for use with naturally infected birds.
Serological Diagnosis of
Brucella
Infections in Odontocetes
Authors: Hernández-Mora Gabriela , Manire Charles A. , González-Barrientos Rocío , Barquero-Calvo Elías , Guzmán-Verri Caterina , Staggs Lydia , Thompson Rachel , Chaves-Olarte Esteban , Moreno Edgardo
Published: April 2009

Brucella ceti
causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used
Brucella melitensis
and
Brucella abortus
lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with
B. ceti
. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (
R
i/g
2
= 0.65 and κ
i/g
= 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (
R
i/c
2
= 0.46,
R
g/c
2
= 0.37 and κ
i/c
= 0.62, κ
g/c
= 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.
Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis
Authors: Scagnolari Carolina , Midulla Fabio , Pierangeli Alessandra , Moretti Corrado , Bonci Enea , Berardi Rosaria , De Angelis Daniela , Selvaggi Carla , Di Marco Paola , Girardi Enrico , Antonelli Guido
Published: April 2009

Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.
HbaR, a 4-Hydroxybenzoate Sensor and FNR-CRP Superfamily Member, Regulates Anaerobic 4-Hydroxybenzoate Degradation by
Rhodopseudomonas palustris
Authors: Egland Paul G. , Harwood Caroline S.
Published: April 2009

Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation. The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by
Rhodopseudomonas palustris
have been identified. Here we describe a regulatory gene,
hbaR
, that is part of the 4-HBA degradation gene cluster. An
hbaR
mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation. The effect of the HbaR protein on expression of
hbaA
encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using
hbaA
::′
lacZ
transcriptional fusions. HbaR was required for a 20-fold induction of β-galactosidase activity that was observed with a chromosomal
hbaA
::′
lacZ
fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA. HbaR also activated expression from a plasmid-borne
hbaA
-′
lacZ
fusion when it was expressed in aerobically grown
Pseudomonas aeruginosa
cells, indicating that the activity of this regulator is not sensitive to oxygen. The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins. It is most closely related to transcriptional activators that are involved in regulating nitrate reduction. Previously, it has been shown that
R. palustris
has an FNR homologue, called AadR, that is also required for 4-HBA degradation. Our evidence indicates that AadR activates expression of
hbaR
in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.
flhDC
, the Flagellar Master Operon of
Xenorhabdus nematophilus
: Requirement for Motility, Lipolysis, Extracellular Hemolysis, and Full Virulence in Insects
Authors: Givaudan Alain , Lanois Anne
Published: April 2009

Xenorhabdus
is a major insect pathogen symbiotically associated with nematodes of the family Steinernematidae. This motile bacterium displays swarming behavior on suitable media, but a spontaneous loss of motility is observed as part of a phenomenon designated phase variation which involves the loss of stationary-phase products active as antibiotics and potential virulence factors. To investigate the role of one of the transcriptional activators of flagellar genes, FlhDC, in motility and virulence, the
Xenorhabdus nematophilus flhDC
locus was identified by functional complementation of an
Escherichia coli flhD
null mutant and DNA sequencing. Construction of
X. nematophilus flhD
null mutants confirmed that the
flhDC
operon controls flagellin expression but also revealed that lipolytic and extracellular hemolysin activity is
flhDC
dependent. We also showed that the
flhD
null mutant displayed a slightly attenuated virulence phenotype in
Spodoptera littoralis
compared to that of the wild-type strain. Thus, these data indicated that motility, lipase, hemolysin, or unknown functions controlled by the
flhDC
operon are involved in the infectious process in insects. Our investigation expands the view of the flagellar regulon as a checkpoint coupled to a major network involving bacterial physiological aspects as well as motility.
Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria
Authors: Bick Julie Ann , Dennis Jonathan J. , Zylstra Gerben J. , Nowack Jason , Leustek Thomas
Published: April 2009

A gene was cloned from
Burkholderia cepacia
DBO1 that is homologous with
Escherichia coli cysH
encoding 3′-phosphoadenylylsulfate (PAPS) reductase. The
B. cepacia
gene is the most recent addition to a growing list of
cysH
homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5′-adenylylsulfate (APS) reductase than they do to
E. coli
CysH. The evidence reported here shows that the
cysH
from one of the species,
Pseudomonas aeruginosa
, encodes APS reductase. It is able to complement an
E. coli cysH
mutant and a
cysC
mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the
cysC
product. Insertional knockout mutation of
P. aeruginosa cysH
produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified
P. aeruginosa
CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 μmol · min
−1
· mg of protein
−1
at pH 8.5 and 30°C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of
cysH
has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.
Cysteine Biosynthesis Pathway in the Archaeon
Methanosarcina barkeri
Encoded by Acquired Bacterial Genes?
Authors: Kitabatake Makoto , So Man Wah , Tumbula Debra L. , Söll Dieter
Published: April 2009

The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic
Escherichia coli
strain NK3 led to the isolation of the
Methanosarcina barkeri cysK
gene [encoding
O
-acetylserine (thiol)-lyase-A], which displays great similarity to bacterial
cysK
genes. Adjacent to
cysK
is an open reading frame orthologous to bacterial
cysE
(serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like
cysM
genes [encoding
O
-acetylserine (thiol)-lyase-B] in
Pyrococcus
spp.,
Sulfolobus solfataricus
, and
Thermoplasma acidophilum
. However, no orthologs for these genes can be found in
Methanococcus jannaschii
,
Methanobacterium thermoautotrophicum
, and
Archaeoglobus fulgidus
, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.
X-Prolyl Dipeptidyl Aminopeptidase Gene (
pepX
) Is Part of the
glnRA
Operon in
Lactobacillus rhamnosus
Authors: Varmanen Pekka , Savijoki Kirsi , Åvall Silja , Palva Airi , Tynkkynen Soile
Published: April 2009

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from
Lactobacillus rhamnosus
1/6 by using the chromogenic substrate
l
-glycyl-
l
-prolyl-β-naphthylamide for screening of a genomic library in
Escherichia coli
. The nucleotide sequence of a 3.5-kb
Hin
dIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from
Lactobacillus helveticus
,
Lactobacillus delbrueckii
, and
Lactococcus lactis
, respectively. By Northern analysis with a
pepX
-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that
pepX
is part of a polycistronic operon in
L. rhamnosus
. Cloning and sequencing of the upstream region of
pepX
revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the
pepX
region was located 25 nucleotides upstream of
glnR
. Northern analysis with
glnA
- and
pepX
-specific probes indicated that transcription from
glnR
promoter results in a 2.0-kb dicistronic
glnR-glnA
transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The
glnA
gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of
glnA
. In the disruption mutant,
glnRA
-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the
glnA
- or
pepX
-specific probe, demonstrating that
pepX
is indeed part of
glnRA
operon in
L. rhamnosus
. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of
pepX
in the
glnA-pepX
intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other
L. rhamnosus
-related entries in data banks.
Genetics of
l
-Sorbose Transport and Metabolism in
Lactobacillus casei
Authors: Yebra María J. , Veyrat Ana , Santos Mario A. , Pérez-Martínez Gaspar
Published: April 2009

Genes encoding
l
-sorbose metabolism of
Lactobacillus casei
ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (
sorRE
) showed high similarity to the transcriptional regulator and the
l
-sorbose-1-phosphate reductase of the sorbose (
sor
) operon from
Klebsiella pneumoniae
. The other genes are transcribed as one unit (
sorFABCDG
) in opposite direction to
sorRE
. The deduced peptide sequence of
sorF
showed homology with the
d
-sorbitol-6-phosphate dehydrogenase encoded in the
sor
operon from
K. pneumoniae
and
sorABCD
to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent
l
-sorbose PTS from
K. pneumoniae
. Finally, the deduced amino acid sequence of a truncated gene (
sorG
) located downstream of
sorD
presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters,
sorRE
and
sorFABCDG
, are induced by
l
-sorbose and subject to catabolite repression by
d
-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in
L. casei
wild-type and
sorBC
mutant strains indicated that
l
-sorbose is taken up by
l
-sorbose-specific enzyme II and that
L. casei
contains an inducible
d
-fructose-specific PTS. Results of growth analysis of those strains and a
man sorBC
double mutant suggested that
l
-sorbose is probably also transported by the
d
-mannose PTS. We also present evidence, from studies on a
sorR
mutant, suggesting that the
sorR
gene encodes a positive regulator of the two
sor
operons. Sequence alignment of SorR, SorC (
K. pneumoniae
), and DeoR (
Bacillus subtilis
) revealed that they might constitute a new group of transcriptional regulators.
Straight and Curved Conformations of FtsZ Are Regulated by GTP Hydrolysis
Authors: Lu Chunlin , Reedy Mary , Erickson Harold P.
Published: April 2009

FtsZ assembles in vitro into protofilaments that can adopt two conformations—the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.
Involvement of Carbon Source and Acetyl Phosphate in the External-pH-Dependent Expression of Porin Genes in
Escherichia coli
Authors: Heyde Martine , Laloi Patrick , Portalier Raymond
Published: April 2009

The porin composition of the
Escherichia coli
cell envelope was analyzed during growth at different external pHs (pHo) as a function of the acetyl phosphate (AcP) level (Δ
ackA pta
or
ackA
mutant, pyruvate or glucose as the carbon source) in the presence or absence of EnvZ. Our results indicate that the AcP level is influenced by the pHo, leading to modulation of the amount of OmpR-P and subsequent pHo-dependent expression of
ompF
and
ompC
. We also propose the existence of a specific signal, independent of EnvZ and AcP, leading to OmpR phosphorylation in response to pyruvate.
Glycine Betaine Transport in
Lactococcus lactis
Is Osmotically Regulated at the Level of Expression and Translocation Activity
Authors: van der Heide Tiemen , Poolman Bert
Published: April 2009

Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that
Lactococcus lactis
uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438–5444, 1993), is osmotically regulated at the levels of both expression and transport activity.
Uptake of 2-Oxoglutarate in
Synechococcus
Strains Transformed with the
Escherichia coli kgtP
Gene
Authors: Vázquez-Bermúdez María Félix , Herrero Antonia , Flores Enrique
Published: April 2009

A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-
14
C]oxoglutarate.
Synechococcus
sp. strain PCC 7942 was transformed with DNA constructs carrying the
Escherichia coli kgtP
gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The
Synechococcus
sp. strains bearing the
kgtP
gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-
14
C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of a
Synechococcus
sp. strain bearing the
kgtP
gene.
Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances
Authors: Strainic Michael G. , Sullivan Jennifer J. , Collado-Vides Julio , deHaseth Pieter L.
Published: April 2009

The p
R
and p
RM
promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p
R
interfered with open complex formation at p
RM
and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p
RM
promoter. A model to explain these observations is proposed. A search of known
Escherichia coli
promoters identified three pairs of divergent promoters with similar separations to those investigated here.
Inorganic Polyphosphate Is Required for Motility of Bacterial Pathogens
Authors: Rashid M. Harunur , Rao Narayana N. , Kornberg Arthur
Published: April 2009

The
ppk
gene encodes polyphosphate kinase (PPK), the principal enzyme in many bacteria responsible for the synthesis of inorganic polyphosphate (polyP) from ATP. A null mutation in the
ppk
gene of six bacterial pathogens renders them greatly impaired in motility on semisolid agar plates; this defect can be corrected by the introduction of
ppk
gene in
trans
. In view of the fact that the motility of pathogens is essential to invade and establish systemic infections in host cells, this impairment in motility suggests a crucial and essential role of PPK or polyP in bacterial pathogenesis.
Identification of SoxS-Regulated Genes in
Salmonella enterica
Serovar Typhimurium
Authors: Pomposiello Pablo J. , Demple Bruce
Published: April 2009

Salmonella enterica
serovar Typhimurium responds to superoxide-generating agents through
soxR
-mediated activation of the
soxS
gene, whose product, SoxS, is necessary for resistance to oxidative stress. The
S. enterica
serovar Typhimurium
soxRS
system also mediates redox-inducible resistance to diverse antibiotics, which may be relevant to clinical infections. In order to identify SoxS-regulated genes in
S. enterica
serovar Typhimurium, a
lacI
-regulated expression system for the
S. enterica
serovar Typhimurium
soxS
gene was developed. This system was used to demonstrate that
soxS
expression is sufficient for the induction of resistance to the superoxide-generating drug paraquat and for the transcriptional activation of the
sodA
and
micF
genes. In addition, a library of random
lacZ
insertions was generated and screened for clones displaying differential β-galactosidase activity in the presence or absence of SoxS. This selection yielded six independent chromosomal
lacZ
transcriptional fusions that were activated by either artificial expression of SoxS or exposure of wild-type cells to micromolar concentrations of paraquat. Moreover, disruption of the inducible genes by the insertions rendered
S. enterica
serovar Typhimurium hypersensitive to millimolar concentrations of paraquat. Nucleotide sequence determination identified the disrupted genes as
sodA
(Mn-containing superoxide dismutase),
fpr
(NADPH:ferredoxin oxidoreductase), and
ydbK
(a putative Fe-S-containing reductase).
Metabolite Repression and Inducer Exclusion in the Proline Utilization Gene Cluster of
Aspergillus nidulans
Authors: Cubero Beatriz , Gómez Dennis , Scazzocchio Claudio
Published: April 2009

The clustered
prnB
,
prnC
, and
prnD
genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in
cis
only on the permease gene (
prnB
) while derepression of
prnD
and
prnC
is largely the result of reversal of inducer exclusion.
Reduced Flux through the Purine Biosynthetic Pathway Results in an Increased Requirement for Coenzyme A in Thiamine Synthesis in
Salmonella enterica
Serovar Typhimurium
Authors: Frodyma Michael , Rubio Aileen , Downs D. M.
Published: April 2009

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in
Salmonella enterica
serovar Typhimurium. Prior work showed that
panE
mutants (
panE
encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of
panE
mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.
A
celR
Mutation Affecting Transcription of Cellulase Genes in
Thermobifida fusca
Authors: Spiridonov Nikolay A. , Wilson David B.
Published: April 2009

Biosynthesis of extracellular cellulases in the cellulose-degrading actinomycete
Thermobifida fusca
is controlled by a transcriptional regulator, CelR, and cellobiose, which acts as an inducer interfering with the CelR-DNA interaction. We report the identification and characterization of a mutation in the
celR
gene that changes Ala
55
in the hinge helix of CelR to Thr. The wild-type and mutant
celR
genes were cloned in
Escherichia coli
, and their protein products were characterized. The CelR mutant protein bound DNA more weakly than the wild-type protein and formed a less stable complex with DNA in the presence of cellobiose. The results of Western analysis and gel retardation experiments suggest that CelR is produced constitutively and its DNA-binding activity is regulated through posttranslational modification.
Identification and Characterization of an Active Plasmid Partition Mechanism for the Novel
Lactococcus lactis
Plasmid pCI2000
Authors: Kearney Karen , Fitzgerald Gerald F. , Seegers Jos F. M. L.
Published: April 2009

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb
Eco
RI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated
repA
, is indeed required for replication. The results also suggest that the origin of replication is located outside
repA
. Upstream and divergently transcribed from
repA
, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated
parA
, is required for stable inheritance of pCI2000 and is active in
trans
. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.
Light-Dependent Regulation of Cyanobacterial Phytochrome Expression
Authors: García-Domínguez M. , Muro-Pastor M. I. , Reyes J. C. , Florencio F. J.
Published: April 2009

A histidine kinase protein (Cph1) with sequence homology and spectral characteristics very similar to those of the plant phytochrome has been recently identified in the cyanobacterium
Synechocystis
sp. strain PCC 6803. Cph1 together with Rcp1 (a protein homologue to the response regulator CheY) forms a light-regulated two-component system whose function is presently unknown. Levels of
cph1 rcp1
mRNA increase in the dark and decrease upon reillumination. A dark-mediated increase in
cph1 rcp1
mRNA levels was inhibited by the presence of glucose, but not by inhibition of the photosynthetic electron flow. The half-life of
cph1 rcp1
transcript in the light was about fourfold shorter than in the dark, indicating that control of
cph1 rcp1
transcript stability is one of the mechanisms by which light regulates expression of the cyanobacterial phytochrome. After 15 min of darkness, 3-min pulses of red, blue, green, and far-red light were equally efficient in decreasing the
cph1 rcp1
mRNA levels. Red light downregulation was not reversed by far-red light, suggesting that
cph1 rcp1
mRNA levels are not controlled by a phytochrome-like photoreceptor. Furthermore, a
Synechocystis
strain containing an H538R Cph1 point mutation, unable to phosphorylate Rcp1, shows normal light-dark regulation of the
cph1 rcp1
transcript levels. Our data suggest a role of cyanobacterial phytochrome in the control of processes required for adaptation in light-dark and dark-light transitions.
The VirR Response Regulator from
Clostridium perfringens
Binds Independently to Two Imperfect Direct Repeats Located Upstream of the
pfoA
Promoter
Authors: Cheung Jackie K. , Rood Julian I.
Published: April 2009

Regulation of toxin production in the gram-positive anaerobe
Clostridium perfringens
occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the
virS
gene, while its cognate response regulator is encoded by the
virR
gene. We have constructed a VirR expression plasmid in
Escherichia coli
and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the
pfoA
gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated
plc
,
colA
, and
pfoR
genes, which encode alpha-toxin, collagenase, and a putative
pfoA
regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the
pfoA
promoter. When this region was deleted, VirR was no longer able to bind to the
pfoA
promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the
pfoA
promoter and activating transcription.
Role of the Tetraheme Cytochrome CymA in Anaerobic Electron Transport in Cells of
Shewanella putrefaciens
MR-1 with Normal Levels of Menaquinone
Authors: Myers Judith M. , Myers Charles R.
Published: April 2009

Shewanella putrefaciens
MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome
c
. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the
cymA
gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a
cymA
knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the
cymA
transcript and CymA protein was also restored, by complementation of MR1-CYMA with the
cymA
gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.
Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of
Saccharomyces cerevisiae
Authors: Avery Simon V. , Malkapuram Srividya , Mateus Carolina , Babb Kimberly S.
Published: April 2009

Saccharomyces cerevisiae
, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of
SOD1
(encoding Cu/Zn superoxide dismutase) rendered
S. cerevisiae
hypersensitive to oxytetracycline (OTC): a
sod1Δ
mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 μg ml
−1
, whereas concentrations of up to 1,000 μg ml
−1
had no effect on the growth of the wild type. OTC resistance was restored in the
sod1
Δ mutant by complementation with wild-type
SOD1
. The effect of OTC appeared to be cytotoxic and was not evident in a
ctt1
Δ (cytosolic catalase) mutant or in the presence of tetracycline.
SOD1
transcription was not induced by OTC, suggesting that constitutive
SOD1
expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and
sod1
Δ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the
sod1
Δ mutant, but not the wild type, to OTC. We propose that Sod1p protects
S. cerevisiae
against a mode of OTC action that is dependent on oxidative damage.
Characterization of the Endogenous Plasmid from
Pseudomonas alcaligenes
NCIB 9867: DNA Sequence and Mechanism of Transfer
Authors: Kwong Stephen M. , Yeo Chew Chieng , Suwanto Antonius , Poh Chit Laa
Published: April 2009

The endogenous plasmid pRA2 from
Pseudomonas alcaligenes
NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The
Pac
25I restriction-modification system and two mobile elements, Tn
5563
and IS
1633
, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the ΩStr
r
/Spc
r
gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of
P. alcaligenes
NCIB 9867 were observed at high frequencies (2.4 × 10
−4
per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into
Pseudomonas putida
RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in
trans
. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host,
P. alcaligenes
NCIB 9867, as well as in
P. putida
RA713 after 100 generations of nonselective growth. Disruption of the pRA2
pac25I
restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.
Proline Catabolism byPseudomonas putida: Cloning, Characterization, and Expression of theputGenes in the Presence of Root Exudates
Authors: Vílchez Susana , Molina Lázaro , Ramos Cayo , Ramos Juan L.
Published: April 2009

Pseudomonas putidaKT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. AP. putidamutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5–Km mutagenesis. The mini-Tn5insertion was located at theputAgene, which is adjacent to and divergent from theputPgene. TheputAgene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein ofEscherichia coli,Salmonella entericaserovar Typhimurium,Rhodobacter capsulatus, and severalRhizobiumstrains. The central part ofP. putidaPutA showed homology to the proline dehydrogenase ofSaccharomyces cerevisiaeandDrosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase ofS. cerevisiaeand a number of aldehyde dehydrogenases. This suggests that inP. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. TheputPgene was homologous to theputPgenes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In aP. putida putA-deficient background, expression of bothputAandputPgenes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced theP. putida putgenes, as determined by using fusions of theputpromoters to ′lacZ. The induction ratio for theputApromoter (about 20-fold) was 6-fold higher than the induction ratio for theputPpromoter.
Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon
Sulfolobus solfataricus
Authors: Hemmi Hisashi , Ikejiri Satoru , Yamashita Satoshi , Nishino Tokuzo
Published: April 2009

Two open reading frames which encode the homologues of (all-
E
) prenyl diphosphate synthase are found in the whole-genome sequence of
Sulfolobus solfataricus
, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in
Escherichia coli
cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg
2+
and Triton X-100 for full activity. It catalyzes consecutive
E
-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.
The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis inSalmonella entericaSerovar Typhimurium
Authors: Tomoyasu Toshifumi , Ohkishi Tomiko , Ukyo Yoshifumi , Tokumitsu Akane , Takaya Akiko , Suzuki Masato , Sekiya Kachiko , Matsui Hidenori , Kutsukake Kazuhiro , Yamamoto Tomoko
Published: April 2009

The ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease inSalmonella entericaserovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis. Cells deleted for ClpXP show “hyperflagellate phenotype,” exhibit overproduction of the flagellar protein, and show a fourfold increase in the rate of transcription of thefliCencoding flagellar filament. The assay for promoter activity of the genes responsible for expression of thefliCshowed that the depletion of ClpXP results in dramatic enhancement of the expression of thefliAencoding sigma factor ς28, leaving the expression level of theflhDmaster operon lying at the top of the transcription hierarchy of flagellar regulon almost normal. These results suggest that the ClpXP may be responsible for repressing the expression of flagellar regulon through the control of the FlhD/FlhC master regulators at the posttranscriptional and/or posttranslational levels. Proteome analysis of proteins secreted from the mutant cells deficient forflhDCandclpXPgenes demonstrated that the ΔflhDmutation abolished the enhanced effect by ΔclpXPmutation on the production of flagellar proteins, suggesting that the ClpXP possibly defines a regulatory pathway affecting the expression of flagellar regulon that is dependent on FlhD/FlhC master regulators.