bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycoinositolphospholipids fromTrypanosoma cruziInterfere with Macrophages and Dendritic Cell Responses
Authors: Brodskyn Claudia , Patricio Julie , Oliveira Rubem , Lobo Lucas , Arnholdt Andrea , Mendonça-Previato Lucia , Barral Aldina , Barral-Netto Manoel
Published: July 2002

To investigate the possible effects of glycoinositolphospholipid (GIPL) fromTrypanosoma cruzion human antigen presenting cells, we tested their effects on lipopolysaccharide (LPS)-stimulated human macrophages and dendritic cells (DC). Human macrophages or DC were incubated with GIPL (50 μg/ml) and LPS (500 pg/ml) and tumor necrosis factor alpha (TNF-α), interleukin 8 (IL-8), IL-10, and IL-12p40 levels in supernatants were analyzed by enzyme-linked immunosorbent assay. TNF-α, IL-10, and IL-12 secretion were significantly decreased by GIPL both in macrophages and DC. In contrast, GIPL did not alter IL-8 production. We also analyzed the expression of CD80, CD86, HLA-DR, CD40, and CD57 on the macrophage surface after stimulation with LPS in the presence or absence ofT. cruziGIPL. GIPL led to a down-regulation in the expression of all tested molecules. We additionally examined the influence ofT. cruziGIPL on the response of human DC to LPS. LPS-induced HLA-DR, CD83, and CD86 up-regulation was significantly inhibited by GIPL. A slight down-regulation in CD80 and CD40 expression on DC surfaces in the presence of GIPL was also noticed. Similarly, GIPL led to down-modulation of CD83, CD80, CD86, and HLA-DR surface expression and TNF-α and IL-10 production when DC were stimulated by CD40L. The ceramide portion of GIPL was responsible for most of the activity exhibited by the whole molecule. Considering the important role of the immune response in determining the fate of the host-parasite relationship, the immunoregulatory activities ofT. cruziGIPL are potentially important for parasite evasion and then pathogenesis of infection with protozoan parasites.
Characterization of the
relA
Gene of
Porphyromonas gingivalis
Authors: Sen Keya , Hayashi Jun-ichiro , Kuramitsu Howard K.
Published: July 2002

Based upon the nucleotide sequence of the
relA
gene from
Escherichia coli
, a gene fragment corresponding to the homologous gene from the pathogenic oral bacterium
Porphyromonas gingivalis
381 was isolated by PCR and utilized to construct a
relA
mutant. The mutant, KS7, was defective in ribosome-mediated ppGpp formation and also in the stringent response.
Inactivation of the
inhA
-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of
Mycobacterium smegmatis
Authors: Vilchèze Catherine , Morbidoni Hector R. , Weisbrod Torin R. , Iwamoto Hiroyuki , Kuo Mack , Sacchettini James C. , Jacobs William R.
Published: July 2002

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (
katG
,
inhA
,
ahpC
,
kasA
, and
ndh
) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates
inhA
, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of
Mycobacterium tuberculosis
causes the accumulation of hexacosanoic acid (C
26:0
), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the
inhA
gene of
Mycobacterium smegmatis
that rendered InhA inactive at 42°C. Thermal inactivation of InhA in
M. smegmatis
resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C
16:0
) and a concomitant increase of tetracosanoic acid (C
24:0
) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of
Mycobacterium bovis
BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C
16:0
, and a concomitant accumulation of C
26:0
. Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH.
A
soxA
Gene, Encoding a Diheme Cytochrome
c
, and a
sox
Locus, Essential for Sulfur Oxidation in a New Sulfur Lithotrophic Bacterium
Authors: Mukhopadhyaya Pratap N. , Deb Chirajyoti , Lahiri Chandrajit , Roy Pradosh
Published: July 2002

A mobilizable suicide vector, pSUP5011, was used to introduce Tn
5-mob
in a new facultative sulfur lithotrophic bacterium, KCT001, to generate mutants defective in sulfur oxidation (Sox
−
). The Sox
−
mutants were unable to oxidize thiosulfate while grown mixotrophically in the presence of thiosulfate and succinate. The mutants were also impaired in oxidizing other reduced sulfur compounds and elemental sulfur as evident from the study of substrate oxidation by the whole cells. Sulfite oxidase activity was significantly diminished in the cell extracts of all the mutants. A
soxA
gene was identified from the transposon-adjacent genomic DNA of a Sox
−
mutant strain. The sequence analysis revealed that the
soxA
open reading frame (ORF) is preceded by a potential ribosome binding site and promoter region with −10- and −35-like sequences. The deduced nucleotide sequence of the
soxA
gene was predicted to code for a protein of 286 amino acids. It had a signal peptide of 26 N-terminal amino acids. The amino acid sequence showed similarity with a putative gene product of
Aquifex aeolicus
, soluble cytochrome
c
551
of
Chlorobium limicola
, and the available partial SoxA sequence of
Paracoccus denitrificans
. The
soxA
-encoded product seems to be a diheme cytochrome
c
for KCT001 and
A. aeolicus
, but the amino acid sequence of
C. limicola
cytochrome
c
551
revealed a single heme-binding region. Another transposon insertion mutation was mapped within the
soxA
ORF. Four other independent transposon insertion mutations were mapped in the 4.4-kb
soxA
contiguous genomic DNA region. The results thus suggest that a
sox
locus of KCT001, essential for sulfur oxidation, was affected by all these six independent insertion mutations.
Phosphorylation-Induced Signal Propagation in the Response Regulator NtrC
Authors: Lee Jonghui , Owens Jeffrey T. , Hwang Ingyu , Meares Claude , Kustu Sydney
Published: July 2002

The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the ς
54
holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrC
D86C
, in which the unique cysteine lies near the top of α-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that α-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede—and may give rise to—the oligomerization needed for transcriptional activation.
Mutations in Multidrug Efflux Homologs, Sugar Isomerases, and Antimicrobial Biosynthesis Genes Differentially Elevate Activity of the ς
X
and ς
W
Factors in
Bacillus subtilis
Authors: Turner Mark S. , Helmann John D.
Published: July 2002

The ς
X
and ς
W
extracytoplasmic function sigma factors regulate more than 40 genes in
Bacillus subtilis
. ς
W
activates genes which function in detoxification and the production of antimicrobial compounds, while ς
X
activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate ς
W
or ς
X
as judged by up-regulation from the autoregulatory promoter site P
W
or P
X
. Fourteen insertions that activate P
W
were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (
yqgE
and
yulE
), a component of the oligopeptide uptake system (
oppA
), and two transmembrane proteins with weak similarity to transporters (
yhdP
and
yueF
). Expression from P
W
is also elevated as a result of inactivation of at least one member of the ς
W
regulon (
ysdB
), an ArsR homolog (
yvbA
), a predicted rhamnose isomerase (
yulE
), and a gene (
pksR
) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate P
X
. Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (
yitG
), a mannose-6-phosphate isomerase gene (
yjdE
), and loci involved in antibiotic synthesis (
srfAB
and possibly
yogA
and
yngK
). Significantly, most insertions that activate P
W
have little or no effect on P
X
, and conversely, insertions that activate P
X
have no effect on P
W
. This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.
Transcriptional and Translational Regulation of α-Acetolactate Decarboxylase of
Lactococcus lactis
subsp.
lactis
Authors: Goupil-Feuillerat Nathalie , Corthier Gérard , Godon Jean-Jacques , Ehrlich S. Dusko , Renault Pierre
Published: July 2002

The α-acetolactate decarboxylase (ALDC) gene,
aldB
, is the penultimate gene of the
leu-ilv-ald
operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in
Lactococcus lactis
. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of α-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the
leu
and
ilv
genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although
aldB
is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The
aldB
ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the
aldB
RBS was responsible for a 100-fold decrease in the level of
aldB
translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of
aldB
translation. Finally, the translation of the
ilvA
gene terminating in the latter structure decreased the level of translation of
aldB
fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the
ilvA
product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.
In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of
Pseudomonas
sp. Strain KWI-56 Lipase
Authors: Yang Junhao , Kobayashi Koei , Iwasaki Yugo , Nakano Hideo , Yamane Tsuneo
Published: July 2002

The expression of lipase from
Pseudomonas
sp. strain KWI-56 (recently reclassified as
Burkholderia cepacia
) had been found to be dependent on an activator gene (
act
) downstream of its structural gene (
lip
). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free
Escherichia coli
S30 coupled transcription-translation system by expressing a recombinant lipase gene (r
lip
) encoding the mature lipase in the presence of its purified activator or by coexpression of r
lip
and
act
. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca
2+
binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca
2+
binding site in the activation of the lipase is also discussed.
Characterization of an HPr Kinase Mutant of
Staphylococcus xylosus
Authors: Huynh Phuong Lan , Jankovic Ivana , Schnell Norbert F. , Brückner Reinhold
Published: July 2002

The
Staphylococcus xylosus
gene
hprK
, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His
6
fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of
S. xylosus
resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of
S. xylosus
. The
hprK
mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of
S. xylosus
according to the availability of preferred carbon sources.
Characterization of the
uup
Locus and Its Role in Transposon Excisions and Tandem Repeat Deletions in
Escherichia coli
Authors: Reddy Manjula , Gowrishankar J.
Published: July 2002

Null mutations in the
Escherichia coli uup
locus (at 21.8 min) serve to increase the frequency of RecA-independent precise excision of transposable elements such as Tn
10
and to reduce the plaque size of bacteriophage Mu (Uup
−
phenotype). By the combined approaches of physical mapping of the mutations, complementation analyses, and protein overexpression from cloned gene fragments, we have demonstrated in this study that the Uup
−
phenotype is the consequence of the absence of expression of the downstream gene (
uup
) of a two-gene operon, caused either directly by insertions in
uup
or indirectly by the polar effect of insertions in the upstream gene (
ycbY
). The promoter for
uup
was mapped upstream of
ycbY
by primer extension analysis on cellular RNA, and assays of reporter gene expression indicated that it is a moderately active, constitutive promoter. The
uup
mutations were also shown to increase, in a RecA-independent manner, the frequencies of nearly precise excision of Tn
10
derivatives and of the deletion of one copy of a chromosomal tandem repeat, suggesting the existence of a shared step or intermediate in the pathways of these latter events and that of precise excision. Finally, we found that mutations that increase the frequency of precise excision of Tn
10
are divisible into two categories, depending upon whether they did (
uup
,
ssb
,
polA
, and
topA
) or did not (
mutHLS
,
dam
, and
uvrD
) also increase precise excision frequency of the mini-Tn
10
derivatives. It is suggested that the differential response of mini-Tn
10
and Tn
10
to the second category of mutations is related to the presence, respectively, of perfect and of imperfect terminal inverted repeats in them.
trans
-Acting Mutations in Loci Other than
kdpDE
That Affect
kdp
Operon Regulation in
Escherichia coli
: Effects of Cytoplasmic Thiol Oxidation Status and Nucleoid Protein H-NS on
kdp
Expression
Authors: Sardesai Abhijit A. , Gowrishankar J.
Published: July 2002

Transcription of the K
+
transport operon
kdp
in
Escherichia coli
is induced during K
+
-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for new
dke
(named
dke
for decreased
kdp
expression) mutations (in loci other than
kdpDE
) that led to substantially decreased
kdp
expression. One
dke
mutation was shown to be in
hns
, encoding the nucleoid protein H-NS. Another
dke
mutation was mapped to
trxB
(encoding thioredoxin reductase), and an equivalent reduction in
kdp
expression was demonstrated also for
trxA
mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the
kdp
expression defect in
trxB
but not
trxA
mutants. Neither
trxB
nor
trxA
affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes
dsbC
and
dsbD
did not affect
kdp
expression, suggesting that the
trx
effects on
kdp
are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced
kdp
expression was observed even in a
trxB
strain that harbored a variant KdpD polypeptide bearing no Cys residues. A
trxB hns
double mutant was even more severely affected for
kdp
expression than either single mutant. The
dke
mutations themselves had no effect on strength of the signal controlling
kdp
expression, and constitutive mutations in
kdpDE
were epistatic to
hns
and
trxB
. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal for
kdp
transcription.
trans
-3-Chloroacrylic Acid Dehalogenase from
Pseudomonas pavonaceae
170 Shares Structural and Mechanistic Similarities with 4-Oxalocrotonate Tautomerase
Authors: Poelarends Gerrit J. , Saunier Raymond , Janssen Dick B.
Published: July 2002

The genes (
caaD1
and
caaD2
) encoding the
trans
-3-chloroacrylic acid dehalogenase (CaaD) of the 1,3-dichloropropene-utilizing bacterium
Pseudomonas pavonaceae
170 were cloned and heterologously expressed in
Escherichia coli
and
Pseudomonas
sp. strain GJ1. CaaD is a protein of 50 kDa that is composed of α-subunits of 75 amino acid residues and β-subunits of 70 residues. It catalyzes the hydrolytic cleavage of the β-vinylic carbon-chlorine bond in
trans
-3-chloroacrylic acid with a turnover number of 6.4 s
−1
. On the basis of sequence similarity, oligomeric structure, and subunit size, CaaD appears to be related to 4-oxalocrotonate tautomerase (4-OT). This tautomerase consists of six identical subunits of 62 amino acid residues and catalyzes the isomerization of 2-oxo-4-hexene-1,6-dioate, via hydroxymuconate, to yield 2-oxo-3-hexene-1,6-dioate. In view of the oligomeric architecture of 4-OT, a trimer of homodimers, CaaD is postulated to be a hexameric protein that functions as a trimer of αβ-dimers. The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the β-subunit and Arg-11 of the α-subunit are active-site residues in CaaD. Pro-1 could act as the proton acceptor/donor, and Arg-11 is probably involved in carboxylate binding. Based on these findings, a novel dehalogenation mechanism is proposed for the CaaD-catalyzed reaction which does not involve the formation of a covalent enzyme-substrate intermediate.
Localization of GerAA and GerAC Germination Proteins in the
Bacillus subtilis
Spore
Authors: Hudson Kaye D. , Corfe Bernard M. , Kemp E. Helen , Feavers Ian M. , Coote Peter J. , Moir Anne
Published: July 2002

The GerAA, -AB, and -AC proteins of the
Bacillus subtilis
spore are required for the germination response to
l
-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the
gerA
genes are found in every spore former so far examined. The
gerA
operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of
B. subtilis
were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5∗, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.
Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases
Authors: van Hylckama Vlieg Johan E. T. , Tang Lixia , Lutje Spelberg Jeffrey H. , Smilda Tim , Poelarends Gerrit J. , Bosma Tjibbe , van Merode Annet E. J. , Fraaije Marco W. , Janssen Dick B.
Published: July 2002

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in
Escherichia coli
, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of
Agrobacterium radiobacter
strain AD1, designated HheC, was purified to homogeneity. The
k
cat
and
K

m

values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s
−1
and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK
a
of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.
Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of
Escherichia coli degP
Mutants and Induces the Cpx and ς
E
Extracytoplasmic Stress Responses
Authors: Langen Geoffrey R. , Harper Jill R. , Silhavy Thomas J. , Howard S. Peter
Published: July 2002

DegP is a periplasmic protease that is a member of both the ς
E
and Cpx extracytoplasmic stress regulons of
Escherichia coli
and is essential for viability at temperatures above 42°C. [U-
14
C]acetate labeling experiments demonstrated that phospholipids were degraded in
degP
mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from
degP
cells at the nonpermissive temperature. A mutation in
pldA
, which encodes outer membrane phospholipase A (OMPLA), was found to rescue
degP
cells from the temperature-sensitive phenotype.
pldA degP
mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants.
degP
and
pldA degP
mutants containing chromosomal
lacZ
fusions to Cpx and ς
E
regulon promoters indicated that both regulons were activated in the
pldA
mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of
degP
mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the
degP
temperature-sensitive phenotype by inducing the Cpx and ς
E
regulons rather than by inactivating the phospholipase per se.
Gene Cluster of
Rhodothermus marinus
High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a
caa
3
-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases
Authors: Santana Margarida , Pereira Manuela M. , Elias Nuno P. , Soares Cláudio M. , Teixeira Miguel
Published: July 2002

The respiratory chain of the thermohalophilic bacterium
Rhodothermus marinus
contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named
rcoxA
to
rcoxD
) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of
rcoxD
. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the
R. marinus
enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E
278
,
Paracoccus denitrificans
numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu
A
domain, that is characteristic of
caa
3
oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this
caa
3
oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the
caa
3
class of heme-copper cytochrome
c
oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.
The C Terminus of ς
32
Is Not Essential for Degradation by FtsH
Authors: Tomoyasu Toshifumi , Arsène Florence , Ogura Teru , Bukau Bernd
Published: July 2002

A key step in the regulation of heat shock genes in
Escherichia coli
is the stress-dependent degradation of the heat shock promoter-specific ς
32
subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including ς
32
, as degradation signals for AAA proteases. We investigated the role of the C terminus of ς
32
in FtsH-dependent degradation by analysis of C-terminally truncated ς
32
mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of ς
32
did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of ς
32
was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated ς
32
mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of ς
32
in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.
Influence of the Nucleoid on Placement of FtsZ and MinE Rings inEscherichia coli
Authors: Sun Qin , Margolin William
Published: July 2002

We previously presented evidence that replicating but unsegregated nucleoids, along with the Min system, act as topological inhibitors to restrict assembly of the FtsZ ring (Z ring) to discrete sites in the cell. To test if nonreplicating nucleoids have similar exclusion effects, we examined Z rings indnaA(temperature sensitive) mutants. Z rings were excluded from centrally localized nucleoids and were often observed at nucleoid edges. Cells with nonreplicating nucleoids formed filaments, some of which contained large nucleoid-free areas in which Z rings were positioned at regular intervals. Because MinE may protect FtsZ from the action of the MinC inhibitor in these nucleoid-free zones, we examined the localization of a MinE-green fluorescent protein (GFP) fusion with respect to Z rings and nucleoids. Like Z rings, MinE-GFP appeared to localize independently of nucleoid position, forming rings at regular intervals in nucleoid-free regions. Unlike FtsZ, however, MinE-GFP often localized on top of nucleoids, replicating or not, suggesting that MinE is relatively insensitive to the nucleoid inhibition effect. These data suggest that both replicating and nonreplicating nucleoids are capable of topologically excluding Z rings but not MinE.
Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from
Streptococcus uberis
Authors: Ward Philip N. , Leigh James A.
Published: July 2002

A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from
Streptococcus uberis
strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of
S. uberis
strains. Analysis of the locus normally occupied by
pauA
revealed an absence of the
pauA
open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the
pauB
gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.
Nucleotide and Amino Acid Sequences of
oriT-traM-traJ-traY-traA-traL
Regions and Mobilization of Virulence Plasmids of
Salmonella enterica
Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium
Authors: Chu Chishih , Chiu Cheng-Hsun , Chu Chi-Hong , Ou Jonathan T.
Published: July 2002

The virulence plasmid of
Salmonella enterica
serovar Gallinarum-Pullorum (pSPV) but not those of
Salmonella enterica
serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the
oriT-traM-traJ-traY-traA-traL
regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the
oriT
of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the
oriT
of the other two pSVs. The nucleotide sequences of the
oriT-traM-traJ-traY-traA-traL
region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in
traJ
,
traY
, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus βαα fragments in the TraY of pSPV and F plasmid, whereas there was only a single βαα fragment in that of pSEV and pSTV.
Automation of Fluorescence-Based PCR for Confirmation of Meningococcal Disease
Authors: Diggle M. A. , Edwards G. F. S. , Clarke S. C.
Published: July 2002

A fluoresence-based PCR method was developed, fully automated, and used to confirm infection with
Neisseria meningitidis
by detection of the meningococcus-specific
ctrA
gene. The method provided a highly sensitive, high-throughput assay that was reproducible and less labor-intensive than manual methods.
Mutations in the
rpoB
Gene of Rifampin-Resistant
Mycobacterium tuberculosis
Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay
Authors: Garcia Lucia , Alonso-Sanz Mercedes , Rebollo Maria J. , Tercero Juan C. , Chaves Fernando
Published: July 2002

Genetic alterations in the
rpoB
gene were characterized in 50 rifampin-resistant (Rif
r
) clinical isolates of
Mycobacterium tuberculosis
complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of
rpoB
mutations was evaluated with isolates of the
M. tuberculosis
complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different
rpoB
mutations among the Rif
r
isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rif
r
allele within each strain, 10 of the 11 Rif
r
genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of
M. tuberculosis
complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rif
r
organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods.
Simple Method for Determining Biovar and Serovar Types of
Ureaplasma urealyticum
Clinical Isolates Using PCR–Single-Strand Conformation Polymorphism Analysis
Authors: Pitcher David , Sillis Margaret , Robertson Janet A.
Published: July 2002

Ureaplasma urealyticum
has been associated with urethritis in men, obstetric problems in women, and respiratory distress syndrome in preterm infants.
U. urealyticum
can be divided into two biovars comprising 14 serovars. Partial sequences of genes encoding the multiple-banded antigens of the cell surface are known. Using a commercially available precast DNA mutation detection gel system, we have developed a simple and reproducible PCR–single-strand conformation polymorphism analysis method for differentiating the biovars of this species that reveals five patterns among the 14 serovars and enables clinical isolates to be typed directly from broth cultures.
Laboratory Diagnosis of
Clostridium difficile
-Associated Diarrhea and Colitis: Usefulness of Premier Cytoclone A+B Enzyme Immunoassay for Combined Detection of Stool Toxins and Toxigenic
C. difficile
Strains
Authors: Lozniewski Alain , Rabaud Christian , Dotto Emmanuelle , Weber Michele , Mory Francine
Published: July 2002

Detection of
Clostridium difficile
toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for
C. difficile
followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of
C. difficile
-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.
Distinguishing
Candida
Species by β-
N
-Acetylhexosaminidase Activity
Authors: Niimi Kyoko , Shepherd Maxwell G. , Cannon Richard D.
Published: July 2002

A variety of fungi produce the hydrolytic enzyme β-
N
-acetylhexosaminidase (HexNAcase), which can be readily detected in assays by using
p
-nitrophenyl-
N
-acetyl-β-
d
-glucosaminide as a substrate. In the present study we developed a microtiter plate-based HexNAcase assay for distinguishing
Candida albicans
and
Candida dubliniensis
strains from other yeast species. HexNAcase activity was detected in 89 of 92 (97%)
C. albicans
strains and 4 of 4
C. dubliniensis
strains but not in 28 strains of eight other
Candida
species, 4
Saccharomyces cerevisiae
strains, or 2
Cryptococcus neoformans
strains. The HexNAcase activity in
C. albicans
and
C. dubliniensis
was strain specific. All except three clinical
C. albicans
isolates among the
C. albicans
strains tested produced enzyme activity within 24 h. These strains did produce enzyme activity, however, after a prolonged incubation period. For two of these atypical strains, genomic DNA at the
C. albicans HEX1
gene locus, which encodes HexNAcase, showed nucleotide differences from the sequence of control strains. Among the other
Candida
species tested, only
C. dubliniensis
had a DNA sequence that hybridized with the
HEX1
probe under low-stringency conditions. The microtiter plate-based assay used in the present study for the detection of HexNAcase activity is a simple, relatively inexpensive method useful for the presumptive identification of
C. albicans
and
C. dubliniensis
.
Molecular Profiles of Group B Streptococcal Surface Protein Antigen Genes: Relationship to Molecular Serotypes
Authors: Kong Fanrong , Gowan Sonia , Martin Diana , James Gregory , Gilbert Gwendolyn L.
Published: July 2002

The study of surface protein antigens of group B streptococci (GBS) is important for understanding of the pathogenesis and epidemiology of infection, and several of these antigens have been proposed as components of GBS conjugate vaccines. In a previous study, we developed a novel PCR-and-sequencing system for identification of GBS serotypes and serosubtypes based on the capsular polysaccharide synthesis (
cps
) gene cluster. In this study, we used published sequences to develop PCR assays for identification of genes encoding GBS surface proteins including C alpha (
bca
), C alpha-like proteins 2 and 3 (
alp2
and
alp3
), Rib (
rib
), and C beta (
bac
). We showed that the prototype R reference strain, Prague 25/60, contained a novel alpha-like protein antigen gene (the proposed
alp4
), which presumably encodes an atypical, but antigenically similar, R-like protein. Initial evaluation of these gene-specific assays showed excellent specificity. By combining
cps
serotypes, serosubtypes, and surface protein gene profiles, we were able to divide 224 GBS isolates into 31 serovariants. GBS
bac-
positive strains could be further subtyped into 11 groups and 20 subgroups. Our results confirmed and extended reported associations between some
cps
serotypes and serosubtypes, on the one hand, and surface protein genes, on the other: serosubtypes III-1 and III-2 were associated with
rib
, serosubtype III-3 with
alp2
, serotype Ib with
bca
and
bac
, and serotype V with
alp3
. The associations between serotype Ia and
bca, bca
repetitive unit, and
bca
repetitive unit-like sequence-containing genes need to be studied further. These PCR-based methods will provide an alternative and objective tool for subtyping of GBS based on surface protein antigen genes.
Comparison of Molecular and Conventional Methods for Typing of Enteroviral Isolates
Authors: Manayani Darly Joseph , Shaji R. V. , Fletcher G. John , Cherian Thomas , Murali N. , Sathish N. , Solomon Tilak , Gnanamuthu Chandran , Sridharan Gopalan
Published: July 2002

Twenty-eight enteroviral isolates obtained from various clinical specimens were typed by Lim-Benyesh-Melnick (LBM) pool-based neutralization, PCR-restriction fragment length polymorphism (RFLP), and partial sequencing of the VP1 region of the enteroviral genome. Sequencing was found to be a good alternative to LBM typing, while PCR-RFLP was inadequate for identification of enteroviral isolates.
Evolution of the IS
6110-
Based Restriction Fragment Length Polymorphism Pattern during the Transmission of
Mycobacterium tuberculosis
Authors: Warren R. M. , van der Spuy G. D. , Richardson M. , Beyers N. , Booysen C. , Behr M. A. , van Helden P. D.
Published: July 2002

Interpretation of the molecular epidemiological data of
Mycobacterium tuberculosis
is dependent on the validity of the assumptions that have been made. It is assumed that the IS
6110
banding pattern is sufficiently stable to define epidemiological events representing ongoing transmission. However, molecular epidemiological data also support the observation that the IS
6110
banding pattern may change over time. Factors affecting this rate may include the nature and duration of disease in a host and the opportunity to experience different host environments during the transmission cycle. To estimate the rate of IS
6110
change occurring during the process of transmission,
M. tuberculosis
isolates from epidemiologically linked patients were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis. The identification of IS
6110
banding pattern changes during ongoing transmission suggested that a rate could be estimated. IS
6110
change was significantly associated with strains with >5 IS
6110
elements (
P
= 0.013) and was not observed in low-copy-number isolates. The minimum rate of appearance of variant strains was calculated to be 0.14 variant cases per source-case per year. This data suggest that clustering of isolates based on identical RFLP patterns is expected to underestimate transmission in patients infected with high-copy-number isolates. A model based on the rate of appearance of both variant and invariant strains demonstrates that the genotypically defined population structure may change by 18.6% during the study period of approximately 6.5 years. The implications for the use of RFLP data for epidemiologic study are discussed.
Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen
Candida parapsilosis
Authors: Nosek Jozef , Tomáška L'ubomír , Ryčovská Adriana , Fukuhara Hiroshi
Published: July 2002

Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen
Candida parapsilosis
. The strategy for identification of
C. parapsilosis
strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy.