bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen Binding and Activation by
Mycoplasma fermentans
Authors: Yavlovich Amichai , Higazi Abd A.-R. , Rottem Shlomo
Published: July 2002

The binding of plasminogen to
Mycoplasma fermentans
was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of
125
I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog ɛ-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one with a dissociation constant (

k
d

) of 0.5 μM and the second with a

k
d

of 7.5 μM. The estimated numbers of plasminogen molecules bound were calculated to be 110 and 790 per cell, respectively. Autoradiograms of ligand blots containing
M. fermentans
membrane proteins incubated with
125
I-labeled plasminogen identified two plasminogen-binding proteins of about 32 and 55 kDa. The binding of plasminogen to
M. fermentans
enhances the activation of plasminogen to plasmin by the urokinase-type plasminogen activator (uPA), as monitored by measuring the breakdown of chromogenic substrate S-2251. Enhancement was more pronounced with the low-molecular-weight and the single-chain uPA variants, known to have low plasminogen activator activities. The binding of plasminogen also promotes the invasion of HeLa cells by
M. fermentans
. Invasion was more pronounced in the presence of uPA, suggesting that the ability of the organism to invade host cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.
Stage-Dependent Role of Nitric Oxide in Control ofTrypanosoma cruziInfection
Authors: Saeftel Michael , Fleischer Bernhard , Hoerauf Achim
Published: July 2002

Trypanosoma cruzi, the causative agent of Chagas' disease, is known to be susceptible to nitric oxide (NO)-dependent killing by gamma interferon-activated macrophages. Mice deficient for inducible nitric oxide synthase (iNOS) are highly susceptible toT. cruzi, and inhibition of iNOS from the beginning of infection was reported to lead to an increase in trypomastigotes in the blood and to high mortality. In the present study, we investigated whether NO production is essential for the control ofT. cruziin all phases of the infection. BALB/c mice were treated at different time intervals afterT. cruziinfection with an iNOS inhibitor, aminoguanidine orl-N6-(1-iminoethyl)-lysine (L-NIL). Treatment initiated with the beginning of the infection resulted in 100% mortality by day 16 postinfection (p.i.). If treatment was started later during the acute phase at the peak of parasitemia (day 20 p.i.), all the mice survived. Parasitemia was cleared and tissue amastigotes became undetectable in these mice even in the presence of the iNOS inhibitor L-NIL. Inhibition of iNOS in the chronic phase of the infection, i.e., from day 60 to day 120 p.i., with L-NIL did not result in a reappearance of parasitemia. These data suggest that while NO is essential forT. cruzicontrol in the early phase of acute infection, it is dispensable in the late acute and chronic phase, revealing a fundamental difference in control mechanisms compared to those in infections by other members of the orderKinetoplastida, e.g.,Leishmania major.
HtrA Homologue of
Legionella pneumophila
: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells
Authors: Pedersen Lisa L. , Radulic Marina , Doric Miljenko , Abu Kwaik Yousef
Published: July 2002

Legionella pneumophila
replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an
L. pneumophila
mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H
103
➤R and S
212
➤A) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the
htrA
mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host
Acanthamoeba polyphaga
. We used a promoterless
lacZ
fusion to the
htrA
promoter to probe the phagosomal microenvironment harboring
L. pneumophila
within macrophages and within
A. polyphaga
for the exposure to stress stimuli. The data show that expression through the
htrA
promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the
htrA
mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed
L. pneumophila
. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the
htrA
mutant, replication of the mutant is not rescued, while replication of a
dotA
mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by
L. pneumophila
(mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.
Disruption of the Genes for ClpXP Protease inSalmonella entericaSerovar Typhimurium Results in Persistent Infection in Mice, and Development of Persistence Requires Endogenous Gamma Interferon and Tumor Necrosis Factor Alpha
Authors: Yamamoto Tomoko , Sashinami Hiroshi , Takaya Akiko , Tomoyasu Toshifumi , Matsui Hidenori , Kikuchi Yuji , Hanawa Tomoko , Kamiya Shigeru , Nakane Akio
Published: July 2002

The enteric pathogenSalmonella entericaserovar Typhimurium, similar to other facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by producing a set of stress proteins that are also induced by various environmental stresses. The stress-induced ClpXP protease is a member of the ATP-dependent proteases, which are known to be responsible for more than 90% of all proteolysis inEscherichia coli. To investigate the contribution of the ClpXP protease to the virulence of serovar Typhimurium we initially cloned theclpPandclpXoperon from the pathogenic strain serovar Typhimurium χ3306 and then created insertional mutations in theclpPand/orclpXgene. The ΔclpPand ΔclpXmutants were used to inoculate BALB/c mice by either the intraperitoneal or the oral route and found to be limited in their ability to colonize organs of the lymphatic system and to cause systemic disease in the host. A variety of experiments were performed to determine the possible reasons for the loss of virulence. An oxygen-dependent killing assay using hydrogen peroxide and paraquat (a superoxide anion generator) and a serum killing assay using murine serum demonstrated that all of the serovar Typhimurium ΔclpPand ΔclpXmutants were as resistant to these killing mechanisms as the wild-type strain. On the other hand, the macrophage survival assay revealed that all these mutants were more sensitive to the intracellular environment than the wild-type strain and were unable to grow or survive within peritoneal macrophages of BALB/c mice. In addition, it was revealed that the serovar Typhimurium ClpXP-depleted mutant was not completely cleared but found to persist at low levels within spleens and livers of mice. Interferon gamma-deficient mice and tumor necrosis factor alpha-deficient mice failed to survive the attenuated serovar Typhimurium infections, suggesting that both endogenous cytokines are essential for regulation of persistent infection with serovar Typhimurium.
P13, an Integral Membrane Protein ofBorrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains
Authors: Noppa Laila , Östberg Yngve , Lavrinovicha Marija , Bergström Sven
Published: July 2002

To elucidate antigens present on the bacterial surface ofBorrelia burgdorferisensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore,p13belongs to a gene family with five additional members inB. burgdorferisensu stricto. Thep13gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of thep13transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis.
Preparation, Immunogenicity, and Protective Efficacy, in a Murine Model, of a Conjugate Vaccine Composed of the Polysaccharide Moiety of the Lipopolysaccharide of
Vibrio cholerae
O139 Bound to Tetanus Toxoid
Authors: Boutonnier Alain , Villeneuve Sylvain , Nato Farida , Dassy Bruno , Fournier Jean-Michel
Published: July 2002

The epidemic and pandemic potential of
Vibrio cholerae
O139 is such that a vaccine against this newly emerged serogroup of
V. cholerae
is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of
V. cholerae
O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from
V. cholerae
O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.
Anaerobic Conditions Induce Expression of Polysaccharide Intercellular Adhesin in
Staphylococcus aureus
and
Staphylococcus epidermidis
Authors: Cramton Sarah E. , Ulrich Martina , Götz Friedrich , Döring Gerd
Published: July 2002

Products of the intercellular adhesion (
ica
) operon in
Staphylococcus aureus
and
Staphylococcus epidermidis
synthesize a linear β-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers
ica
gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both
S. aureus
and
S. epidermidis
, although the regulation is less stringent in
S. epidermidis
. Anaerobiosis also dramatically stimulates
ica
-specific mRNA expression in
ica
- and polysaccharide
-
positive strains of both
S. aureus
and
S. epidermidis.
These data suggest a mechanism whereby
ica
gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.
Long-Term Pertussis-Specific Immunity after Primary Vaccination with a Combined Diphtheria, Tetanus, Tricomponent Acellular Pertussis, and Hepatitis B Vaccine in Comparison with That after Natural Infection
Authors: Esposito Susanna , Agliardi Tiziano , Giammanco Anna , Faldella Giacomo , Cascio Antonio , Bosis Samantha , Friscia Ornella , Clerici Mario , Principi Nicola
Published: July 2002

The aim of this study was to compare pertussis-specific humoral and cellular immunity in children 5 years after a primary vaccination with a combined diphtheria, tetanus, tricomponent acellular pertussis, and hepatitis B vaccine (DTaP-HBV; InfanrixHepB; SmithKline Beecham) with immunity after natural infection. The subjects were 38 children aged 5 to 6 years who received DTaP-HBV at 3, 5, and 11 months of life and 21 subjects of similar ages and sex who acquired pertussis in the first year of life. Immunoglobulin G (IgG) antibody titers against
Bordetella pertussis
antigens, peripheral blood mononuclear cell-specific proliferation, and the secretion of cytokines were evaluated. After 5 years, only a small proportion of vaccinated and infected children had significant specific concentrations of IgG in serum against all three
B. pertussis
antigens, and T-cell responses persisted in a minority of subjects. A preferential type 1 cytokine response with the secretion of gamma interferon was observed in the pertussis group, whereas a type 2 skewed response was observed in the vaccinated children; however, the quantitative differences in the cytokines produced by DTaP-HBV and natural infection were minimal. In conclusion, our results show that the immune responses induced by primary pertussis vaccination are qualitatively and quantitatively similar to those seen in children who recovered from natural infection and highlight the need for booster immunization with pertussis vaccines in order to maintain adequate levels of a specific immune response to
B. pertussis
.
Tandem Repeat Deletion in the Alpha C Protein of Group B
Streptococcus
Is
recA
Independent
Authors: Puopolo Karen M. , Hollingshead Susan K. , Carey Vincent J. , Madoff Lawrence C.
Published: July 2002

Group B streptococci (GBS) contain a family of protective surface proteins characterized by variable numbers of repeating units within the proteins. The prototype alpha C protein of GBS from the type Ia/C strain A909 contains a series of nine identical 246-bp tandem repeat units. We have previously shown that deletions in the tandem repeat region of the alpha C protein affect both the immunogenicity and protective efficacy of the protein in animal models, and these deletions may serve as a virulence mechanism in GBS. The molecular mechanism of tandem repeat deletion is unknown. To determine whether RecA-mediated homologous recombination is involved in this process, we identified, cloned, and sequenced the
recA
gene homologue from GBS. A strain of GBS with
recA
deleted, A909Δ
recA
, was constructed by insertional inactivation in the
recA
locus. A909Δ
recA
demonstrated significant sensitivity to UV light, and the 50% lethal dose of the mutant strain in a mouse intraperitoneal model of sepsis was 20-fold higher than that of the parent strain. The spontaneous rate of tandem repeat deletion in the alpha C protein in vitro, as well as in our mouse model of immune infection, was studied using A909Δ
recA
. We report that tandem repeat deletion in the alpha C protein does occur in the absence of a functional
recA
gene both in vitro and in vivo, indicating that tandem repeat deletion in GBS occurs by a
recA
-independent recombinatorial pathway.
Intimin-Specific Immune Responses Prevent Bacterial Colonization by the Attaching-Effacing PathogenCitrobacter rodentium
Authors: Ghaem-Maghami Marjan , Simmons Cameron P. , Daniell Sarah , Pizza Mariagrazia , Lewis David , Frankel Gad , Dougan Gordon
Published: July 2002

The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC)Escherichia coli,enteropathogenicE. coli(EPEC), and the rodent pathogenCitrobacter rodentium.Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses inC. rodentium-infected mice. Mice infected withC. rodentiumdeveloped robust acquired immunity and were resistant to reinfection with wild-typeC. rodentiumor aC. rodentiumderivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280α from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280α, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-typeC. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388–667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of aC. rodentiuminfection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.
Cryptosporidium parvum
Infection Requires Host Cell Actin Polymerization
Authors: Elliott David A. , Coleman Daniel J. , Lane Michael A. , May Robin C. , Machesky Laura M. , Clark Douglas P.
Published: July 2002

The intracellular protozoan parasite
Cryptosporidium parvum
accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.
Induction of an AIDS Virus-Related Tuberculosis-Like Disease in Macaques: a Model of Simian Immunodeficiency Virus- Mycobacterium Coinfection
Authors: Shen Yun , Zhou Dejiang , Chalifoux Laura , Shen Ling , Simon Meredith , Zeng Xuejun , Lai Xioamin , Li Yunyuan , Sehgal Prabhat , Letvin Norman L. , Chen Zheng W.
Published: July 2002

The mechanism by which human immunodeficiency virus (HIV)-Mycobacterium tuberculosiscoinfection facilitates development of HIV-related tuberculosis is poorly characterized. Macaque models of simian immunodeficiency virus (SIVmac)-Mycobacterium bovisBCG coinfection were employed to explore the pathogenesis of AIDS virus-related tuberculosis. Following BCG coinfection, SIV (SIV)-infected macaques with high viral loads developed an SIV-related tuberculosis-like disease. This disease was characterized clinically by a syndrome of diarrhea, anorexia, weight loss, and altered levels of consciousness and pathologically by the presence of disseminated granulomas. In contrast, SIVmac-infected macaques with low viral loads either showed no evidence of BCG-induced disease or developed focal granulomatous lesions. Pathogenic SIV-BCG interactions appeared to play a critical role in triggering the development of this SIV-related tuberculosis-like disease. BCG coinfection enhanced the destruction of CD4+T cells in SIVmac-infected macaques whose viral loads were high. Reciprocally, exacerbations of SIV disease led to marked suppression of BCG-specific T-cell responses, persistence of the BCG infection, and development of an SIV-related tuberculosis-like disease. Furthermore, development of this SIV-related tuberculosis-like disease was also seen in naïve macaques simultaneously inoculated with SIVmacand BCG. These results provide in vivo evidence that coinfection of AIDS virus-infected individuals with an avirulent mycobacterium can lead to development of a tuberculosis-like disease.
Naturally Attenuated, Orally Administered
Mycobacterium microti
as a Tuberculosis Vaccine Is Better than Subcutaneous
Mycobacterium bovis
BCG
Authors: Manabe Yukari C. , Scott Cherise P. , Bishai William R.
Published: July 2002

Mycobacterium microti
is phylogenetically closely related to
Mycobacterium tuberculosis
and is a member of that complex of organisms. It is a curved, acid-fast bacillus that is naturally attenuated with a narrow host range for
Microtus
species only. In this study, we confirm the unique susceptibility of voles to infection with
M. microti
and the relative resistance of mice with a significantly lower organism burden after 8 weeks of infection. In addition, histopathologic examination of lungs reveals a lack of cellular, granulomatous aggregates characteristically seen in murine
M. tuberculosis
infection. In the past,
M. microti
has been used successfully in humans as a vaccine against tuberculosis but was associated with cutaneous reactions. In an attempt to circumvent this adverse effect, we report the efficacy of aerosol and oral vaccination with
M. microti.
High-dose orogastric vaccination with
M. microti
resulted in a statistically significant improvement in protection against aerosol challenge with virulent
M. tuberculosis
in the murine model compared with subcutaneous
M. bovis
BCG Pasteur vaccination.
Identification of
Escherichia coli
Genes That Are Specifically Expressed in a Murine Model of Septicemic Infection
Authors: Khan Muhammad A. , Isaacson Richard E.
Published: July 2002

Identification and characterization of bacterial genes that are induced during the disease process are important in understanding the molecular mechanism of disease and can be useful in designing antimicrobial drugs to control the disease. The identification of in vivo induced (
ivi
) genes of an
Escherichia coli
septicemia strain by using antibiotic-based in vivo expression technology is described. Bacterial clones resistant to chloramphenicol in vivo were recovered from the livers of infected mice. Most of the
ivi
clones were sensitive to chloramphenicol when grown in vitro. Using reverse transcription-PCR, it was demonstrated that selected
ivi
clones expressed
cat
in the livers of infected mice but not during in vitro growth. A total of 750 colonies were recovered after three successive rounds of in vivo selection, and 168 isolated
ivi
clones were sequenced. The sequence analysis revealed that 37 clones encoded hypothetical proteins found in
E. coli
K-12, whereas 10 clones contained genes that had no significant homology to DNA sequences in GenBank. Two clones were found to contain transposon-related functions. Other clones contained genes required for amino acid metabolism, anaerobic respiration, DNA repair, the heat shock response, and the cellular repressor of the SOS response. In addition, one clone contained the aerobactin biosynthesis gene
iucA.
Mutations were introduced in to seven of the identified
ivi
genes. An in vivo mouse challenge-competition assay was used to determine if the mutants were attenuated. The results suggested that these
ivi
genes were important for survival in vivo, and three of the seven mutant
ivi
clones were required for successful infection of mice.
Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in
Rhodobacter capsulatus
Is Coregulated by the RegB-RegA Two-Component Regulatory System
Authors: Elsen Sylvie , Dischert Wanda , Colbeau Annette , Bauer Carl E.
Published: July 2002

Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H
2
oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in
Rhodobacter capsulatus
. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of
nifA2
, which encodes one of the two functional copies of the
nif
-specific transcriptional activator, NifA. Expression of
nifA2
but not
nifA1
is reduced in the
reg
mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for
nifA2
expression, RegA acts as a coactivator of
nifA2
. We also demonstrated that in
reg
mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H
2
oxidation, as well as the energy-demanding processes of N
2
fixation and CO
2
assimilation.
Prolonged Stationary-Phase Incubation Selects for
lrp
Mutations in
Escherichia coli
K-12
Authors: Zinser Erik R. , Kolter Roberto
Published: July 2002

Evolution by natural selection occurs in cultures of
Escherichia coli
maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of
rpoS
, encoding the stationary-phase global regulator, ς
S
(M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an
rpoS
strain had acquired a GASP mutation in
lrp
. This GASP allele,
lrp-1141
, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The
lrp-1141
allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a
lrp
null allele generated by a transposon insertion has a fitness gain identical to that of the
lrp-1141
allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the
lrp-1141
GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the
lrp
GASP phenotype, and hence the
lrp
GASP phenotype is due to more global physiological changes.
Function of the ς
E
Regulon in Dead-Cell Lysis in Stationary-Phase
Escherichia coli
Authors: Nitta Takeshi , Nagamitsu Hiroshi , Murata Masayuki , Izu Hanae , Yamada Mamoru
Published: July 2002

Elevation of active ς
E
levels in
Escherichia coli
by either repressing the expression of
rseA
encoding an anti-ς
E
factor or cloning
rpoE
in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells,
rpoE
expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase
E. coli
occurs in a ς
E
-dependent fashion. We propose there is a novel physiological function of the ς
E
regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.
Role of
Azotobacter vinelandii mucA
and
mucC
Gene Products in Alginate Production
Authors: Núñez Cinthia , León Renato , Guzmán Josefina , Espı́n Guadalupe , Soberón-Chávez Gloria
Published: July 2002

Azotobacter vinelandii
produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor ς
E
regulates the expression of functions related to the extracytoplasmic compartments. In
A. vinelandii
and
Pseudomonas aeruginosa
, the ς
E
factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the
mucA
,
mucB
,
mucC
, and
mucD
genes. In this work, we studied the transcriptional regulation of the
A. vinelandii algU-mucABCD
gene cluster, as well as the role of the
mucA
and
mucC
gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.
Expression of the Multidrug Resistance Transporter NorA from
Staphylococcus aureus
Is Modified by a Two-Component Regulatory System
Authors: Fournier Bénédicte , Aras Rahul , Hooper David C.
Published: July 2002

To dissect genetically the regulation of NorA, a multidrug transporter of
Staphylococcus aureus
, we analyzed the differential expression of the
norA
promoter using a transcriptional fusion with a β-lactamase reporter gene. Expression studies with an
arlS
mutant revealed that the
norA
promoter is ArlS dependent. The
arlR-arlS
locus was shown to code for a two-component regulatory system. The protein ArlR has strong similarity to response regulators, and ArlS has strong similarity to protein histidine kinases. We have also analyzed the 350-bp region upstream of the Shine-Dalgarno sequence of
norA
by gel mobility shift experiments. It was shown that only the 115-bp region upstream of the promoter was necessary for multiple binding of an 18-kDa protein. From transcriptional fusions, we have localized four different putative boxes of 6 bp, which appear to play a role in the binding of the 18-kDa protein and in the up-regulation of
norA
expression in the presence of the
arlS
mutation. Furthermore, the gel mobility shift of the 18-kDa protein was modified in the presence of the
arlS
mutation, and the
arlS
mutation altered the growth-phase regulation of NorA. These results indicate that expression of
norA
is modified by a two-component regulatory system.
Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from
Rhodococcus erythropolis
Authors: Lichtinger Thomas , Reiss Gila , Benz Roland
Published: July 2002

Organic solvent extracts of whole cells of the gram-positive bacterium
Rhodococcus erythropolis
contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of
R. erythropolis
was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of
R. erythropolis
were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.
PpdD Type IV Pilin of
Escherichia coli
K-12 Can Be Assembled into Pili in
Pseudomonas aeruginosa
Authors: Sauvonnet Nathalie , Gounon Pierre , Pugsley Anthony P.
Published: July 2002

Escherichia coli
K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However,
E. coli
K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putative
E. coli
piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (
ppdD
) and the linked assembly genes
hofB
and
hofC
(homologues of the
Pseudomonas aeruginosa
type IV pilus assembly genes
pilB
and
pilC
) did not lead to pilus production. However, expression of the
ppdD
gene in
P. aeruginosa
led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in
P. aeruginosa
was dependent on the expression of the
pilB
and
pilC
genes and independent of expression of the
P. aeruginosa
pilin structural gene
pilA.
Evolution of Arginine Biosynthesis in the Bacterial Domain: Novel Gene-Enzyme Relationships from Psychrophilic
Moritella
Strains (
Vibrionaceae
) and Evolutionary Significance of
N
-α-Acetyl Ornithinase
Authors: Xu Ying , Liang Ziyuan , Legrain Christianne , Rüger Hans J. , Glansdorff Nicolas
Published: July 2002

In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of
N
-α-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (
argJ
encoded). Only two exceptions had been reported—the
Enterobacteriaceae
and
Myxococcus xanthus
(members of the γ and δ groups of the class
Proteobacteria
, respectively)—in which ornithine is produced from
N
-α-acetylornithine by a deacylase, acetylornithinase (AOase) (
argE
encoded). We have investigated the gene-enzyme relationship in the arginine regulons of two psychrophilic
Moritella
strains belonging to the
Vibrionaceae
, a family phylogenetically related to the
Enterobacteriaceae
. Most of the
arg
genes were found to be clustered in one continuous sequence divergently transcribed in two wings,
argE
and
argCBFGH(A)
[“
H(A)
” indicates that the argininosuccinase gene consists of a part homologous to known
argH
sequences and of a 3′ extension able to complement an
Escherichia coli
mutant deficient in the
argA
gene, encoding
N
-α-acetylglutamate synthetase, the first enzyme committed to the pathway]. Phylogenetic evidence suggests that this new clustering pattern arose in an ancestor common to
Vibrionaceae
and
Enterobacteriaceae
, where OATase was lost and replaced by a deacylase. The AOase and ornithine carbamoyltransferase of these psychrophilic strains both display distinctly cold-adapted activity profiles, providing the first cold-active examples of such enzymes.
Biochemical and Physical Properties of theMethanococcus jannaschii20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome
Authors: Wilson Heather L. , Ou Mark S. , Aldrich Henry C. , Maupin-Furlow Julie
Published: July 2002

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophileMethanococcus jannaschii. In addition, theM. jannaschii20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited byN-ethylmaleimide andp-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively highVmaxfor ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Piper min per mg of protein as well as a relatively low affinity for CTP and ATP withKmvalues of 307 and 497 μM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Δ1–73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that theM. jannaschiias well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.
Membrane Topology of the NixA Nickel Transporter of
Helicobacter pylori
: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III
Authors: Fulkerson John F. , Mobley Harry L. T.
Published: July 2002

NixA, the high-affinity cytoplasmic membrane nickel transport protein of
Helicobacter pylori
, imports Ni
2+
into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with β-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni
2+
transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni
2+
transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.
Two ResD-Controlled Promoters Regulate
ctaA
Expression in
Bacillus subtilis
Authors: Paul Salbi , Zhang Xiaohui , Hulett F. Marion
Published: July 2002

The
Bacillus subtilis
ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration.
ctaA
is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The
ctaAB
-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full
ctaA
promoter activity. In this study, we demonstrate the role of ResD∼P in the activation of the
ctaA
promoter using an in vitro transcription system. The results indicate that the
ctaA
promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by Eς
A
, transcription which was enhanced approximately 1.5-fold by ResD and 5-fold by ResD∼P. The downstream promoter (Ps) required both binding sites A2 and A3 and was not transcribed by Eς
A
with or without ResD∼P. RNA polymerase (RNAP) isolated from
B. subtilis
when cells were at the end of exponential growth (T
0
) or 3, 4, or 5 h into the stationary phase (T
3
, T
4
, or T
5
, respectively) was used in in vitro transcription assays. Maximal transcription from Ps required T
4
RNAP plus ResD∼P. RNAP isolated from a
spo0A
or a
sigE
mutant strain was not capable of Ps transcription. Comparison of the Ps promoter sequence with the SigE binding consensus suggests that the
ctaA
Ps promoter may be a SigE promoter. The collective data from ResD footprinting, in vivo promoter deletion analysis, and in vitro transcription assays suggest that
ctaA
is transcribed during late exponential to early stationary phases of growth from the Pv promoter, which requires ResD binding site A2, Eς
A
, and ResD∼P, and during later stationary phase from Ps, which requires binding sites A2 and A3, ResD∼P, and Eς
E
or a sigma factor whose transcription is dependent on SigE.
Autoregulation of the
dnaA-dnaN
Operon and Effects of DnaA Protein Levels on Replication Initiation in
Bacillus subtilis
Authors: Ogura Yoshitoshi , Imai Yukiho , Ogasawara Naotake , Moriya Shigeki
Published: July 2002

In
Escherichia coli,
the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in
B. subtilis,
we constructed a strain in which a copy of the
dnaA
gene was integrated at the
purA
locus on the chromosome under the control of an isopropyl-β
-d-
thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native
dnaA-dnaN
operon was greatly reduced at high DnaA levels, but it was increased in a
dnaA
-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the
dnaN
gene was added downstream of the additional
dnaA
gene at
purA
, the cells grew at high DnaA levels, suggesting that depletion of DnaN (β subunit of DNA polymerase III) within the cell by repression of the native
dnaA-dnaN
operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in
B. subtilis
.
Succinyl-CoA:(
R
)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria
Authors: Leutwein Christina , Heider Johann
Published: July 2002

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (
R
)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(
R
)-benzylsuccinate CoA-transferase activating (
R
)-benzylsuccinate to the CoA-thioester was purified and characterized from
Thauera aromatica
. The enzyme is fully reversible and forms exclusively the 2-(
R
)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (
R
)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α
2
β
2
conformation. Identity of the subunits with the products of the previously identified toluene-induced
bbsEF
genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (
E
)-cinnamoyl-CoA:(
R
)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates.
Dehalogenation of Dichloromethane by Dichloromethane Dehalogenase/Glutathione
S
-Transferase Leads to Formation of DNA Adducts
Authors: Kayser Martin F. , Vuilleumier Stéphane
Published: July 2002

Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione
S
-transferase was demonstrated. Adducts included dichloromethane carbon and glutathione sulfur atoms. A reaction with DNA occurred preferentially at guanine bases. Increased DNA degradation in a
polA
mutant of
Methylobacterium dichloromethanicum
DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.
Gene Cluster on pAO1 of
Arthrobacter nicotinovorans
Involved in Degradation of the Plant Alkaloid Nicotine: Cloning, Purification, and Characterization of 2,6-Dihydroxypyridine 3-Hydroxylase
Authors: Baitsch Daniel , Sandu Cristinel , Brandsch Roderich , Igloi Gabor L.
Published: July 2002

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of
Arthrobacter nicotinovorans
. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in
Escherichia coli
. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.