bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum S100B Levels in Patients with Lupus Erythematosus: Preliminary Observation
Authors: Portela Luis V. C. , Brenol João C. T. , Walz Roger , Bianchin Marino , Tort Adriano B. L. , Canabarro Ubirajara P. , Beheregaray Simone , Marasca João A. , Xavier Ricardo M. , Neto Eurico C. , Gonçalves Carlos A. , Souza Diogo O.
Published: July 2002

S100B is an astrocytic calcium-binding protein which has been proposed as a biochemical marker of brain damage or dysfunction in acute and chronic diseases. We investigated whether serum S100B levels could be related to systemic lupus erythematosus (SLE) activity. Patients were grouped as having inactive SLE (ISLE), active SLE without central nervous system (CNS) involvement (ASLE), or active SLE with unequivocal neurologic or psychiatric manifestation (NPSLE). The control group consisted of age- and sex-matched healthy blood donors. S100B levels were determined using a luminescence immunoassay. All SLE groups had higher levels of serum S100B than the control group. Among the SLE groups, significantly higher levels of serum S100B protein were found in the NPSLE group than in the ISLE and ASLE groups, and there was no significant difference in S100B levels between the ISLE and ASLE groups. These preliminary results point to a putative relevance of serum S100B protein levels in SLE patients, specifically concerning CNS involvement present in this disease.
Host Cellular Immune Response to Pneumococcal Lung Infection in Mice
Authors: Kadioglu Aras , Gingles Neill A. , Grattan Kate , Kerr Alison , Mitchell Tim J. , Andrew Peter W.
Published: July 2002

Although there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs. We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs. The effect on bacterial growth in the lungs also was assessed. There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline. The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers. The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin. Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci. This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth. Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense. Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs. Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.
Mutations in the Extracellular Protein Secretion Pathway Genes (
eps
) Interfere with Rugose Polysaccharide Production in and Motility of
Vibrio cholerae
Authors: Ali Afsar , Johnson Judith A. , Franco Augusto A. , Metzger Daniel J. , Connell Terry D. , Morris J. Glenn , Sozhamannan Shanmuga
Published: July 2002

Vibrio cholerae
is the causal organism of the diarrheal disease cholera. The rugose variant of
V. cholerae
is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated
eps
) is involved directly or indirectly in the production of rugose polysaccharide. A Tn
phoA
insertion in
epsD
gene of the
eps
operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the
epsD
and
epsE
genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the
eps
system in pathogenesis and environmental survival of
V. cholerae
.
Polymorphism of Repeated Regions of Pertactin in
Bordetella pertussis
,
Bordetella parapertussis
, and
Bordetella bronchiseptica
Authors: Boursaux-Eude Caroline , Guiso Nicole
Published: July 2002

Pertactin is an outer membrane protein expressed by
Bordetella pertussis
,
Bordetella parapertussis
, and
Bordetella bronchiseptica
that induces protective immunity to
Bordetella
infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed that
B. parapertussis
pertactin is invariant, whereas
B. pertussis
pertactin varies mostly in region I and
B. bronchiseptica
pertactin varies in both repeated regions I and II, but mostly in region II. These differences may result from specific characteristics of these
Bordetella
species.
Inhibition of Bacterial Superantigens by Peptides and Antibodies
Authors: Visvanathan Kumar , Charles Alain , Bannan Jason , Pugach Pavel , Kashfi Khosrow , Zabriskie John B.
Published: July 2002

The pyrogenic exotoxins of group A streptococci and staphylococcal enterotoxins are a family of structurally related superantigens with similar biological activity. Two distinct areas have been identified which have a highly conserved amino acid homology in all of the toxin families. A number of peptides were constructed from these regions, some of which were concatenated and polymerized to enhance their immunogenicity in animals. Antibodies prepared against these polymerized peptides were used to serologically identify the majority of the superantigen toxins, block the biological activities of the superantigens, and protect an experimental animal model against shock. In addition certain peptides were able per se to block up to 90% of the proliferative responses induced by the toxins. The peptide also proved protective in a septic shock model in mice. Binding experiments indicate that the peptide binds tightly to the major histocompatibility complex class II molecule, thus preventing binding and hence activation of the superantigen. The selective and rapid binding of the peptide to the major histocompatibility complex class II molecule may lead to a novel therapeutic modality in treatment of superantigen-mediated diseases.
Mosaic Genes and Mosaic Chromosomes: Intra- and Interspecies Genomic Variation of
Streptococcus pneumoniae
Authors: Hakenbeck Regine , Balmelle Nadège , Weber Beate , Gardès Christophe , Keck Wolfgang , de Saizieu Antoine
Published: July 2002

Streptococcus pneumoniae
remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20
S. pneumoniae
isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between
S. pneumoniae
and commensal
Streptococcus mitis
and
Streptococcus oralis
strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.
Antibodies to a Surface-Exposed, N-terminal Domain of Aggregation Substance Are Not Protective in the Rabbit Model of
Enterococcus faecalis
Infective Endocarditis
Authors: McCormick John K. , Hirt Helmut , Waters Christopher M. , Tripp Timothy J. , Dunny Gary M. , Schlievert Patrick M.
Published: July 2002

The aggregation substance (AS) surface protein from
Enterococcus faecalis
has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS
44–331
) was cloned with a C-terminal histidine tag translational fusion and expressed from
Escherichia coli
. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS
44–331
reacted specifically to Asc10 expressed from
E. faecalis
OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS
44–331
bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS
44–331
were opsonic for
E. faecalis
expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS
44–331
were challenged intravenously with
E. faecalis
cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to
E. faecalis
and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.
Human Peripheral Blood T Cells, Monocytes, and Macrophages Secrete Macrophage Inflammatory Proteins 1α and 1β following Stimulation with Heat-InactivatedBrucella abortus
Authors: Zaitseva Marina , King Lisa R. , Manischewitz Jody , Dougan Michael , Stevan Lee , Golding Hana , Golding Basil
Published: July 2002

Heat-killedBrucella abortus(HBa) has been proposed as a carrier for therapeutic vaccines for individuals with immunodeficiency, due to its abilities to induce interleukin-2 (IL-2) and gamma interferon (IFN-γ) in both CD4+and CD8+T cells and to upregulate antigen-presenting cell functions (including IL-12 production). In the current study, we investigated the ability of HBa or lipopolysaccharide isolated from HBa (LPS-Ba) to elicit β-chemokines, known to bind to the human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 and to block viral cell entry. It was found that human peripheral blood mononuclear cells secreted β-chemokines following stimulation with HBa, and this effect could not be blocked by anti-IFN-γ neutralizing antibodies. Among purified T cells, macrophage inflammatory protein 1α and 1β (MIP-1α and MIP-1β, respectively) secretion was observed primarily in human CD8+T cells. The kinetics of β-chemokine induction in T cells were slow (3 to 4 days). The majority of β-chemokine-producing CD8+T cells also produced IFN-γ following HBa stimulation, as determined by triple-color intracellular staining. A significant number of CD8+T cells contained stored MIP-1β that was released after HBa stimulation. Both HBa and LPS-Ba stimulated high levels of MIP-1α and MIP-1β production in elutriated monocytes and even higher levels in macrophages. In these cells, β-chemokine mRNA was upregulated within 30 min and proteins were secreted within 4 h of stimulation. The monocyte- and macrophage-derived β-chemokines were sufficient to block CCR5-dependent HIV-1 envelope-mediated cell fusion. These data suggest that, in addition to the ability of HBa to elicit antigen-specific humoral and cellular immune responses, HBa-conjugated HIV-1 proteins or peptides would also generate innate chemokines with antiviral activity that could limit local viral spread during vaccination in vivo.
Intracellular Survival ofBrucellaspp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes
Authors: Rittig Michael G. , Alvarez-Martinez Maria-Teresa , Porte Françoise , Liautard Jean-Pierre , Rouot Bruno
Published: July 2002

Brucellaspp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated howB. suisandB. melitensisenter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence ofBrucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of allBrucellaorganisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival ofBrucella. These results indicate that (i) human monocytes readily internalizeBrucellain a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of someBrucellaphagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalizedBrucelladepends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.
Porphyromonas gingivalis
Traffics to Autophagosomes in Human Coronary Artery Endothelial Cells
Authors: Dorn Brian R. , Dunn William A. , Progulske-Fox Ann
Published: July 2002

Porphyromonas gingivalis
is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that
P. gingivalis
is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of
P. gingivalis
381 to the autophagic pathway.
P. gingivalis
381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression of
P. gingivalis
381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containing
P. gingivalis
colocalize with Rab5 and HsGsa7p early after internalization. At later times,
P. gingivalis
colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with
P. gingivalis
381. The intracellular survival of
P. gingivalis
381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing
P. gingivalis
381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that
P. gingivalis
381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.
Chlamydia pneumoniaeSecretion of a Protease-Like Activity Factor for Degrading Host Cell Transcription Factors Is Required for Major Histocompatibility Complex Antigen Expression
Authors: Fan Peiyi , Dong Feng , Huang Yanqing , Zhong Guangming
Published: July 2002

Chlamydia pneumoniaeis a causative agent for many respiratory infections and has been associated with cardiovascular diseases in humans. The pathogenicity ofC. pneumoniaeis thought to depend on its ability to cause persistent infection and to evade host defense. Genome sequence analysis indicates thatC. pneumoniaeencodes a homologue of a chlamydial protease-like activity factor fromC. trachomatis(CPAFct). We designated theC. pneumoniaehomologue as CPAFcp. Recombinant CPAFcp was produced and found to degrade RFX5, a host transcription factor required for major histocompatibility complex (MHC) antigen expression. The degradation was inhibitable by lactacystin, an irreversible proteasome inhibitor. Furthermore, CPAFcp was secreted into host cytosol byC. pneumoniaeorganisms. Depletion of theC. pneumoniae-secreted CPAFcp with specific antibodies completely ablated the RFX5 degradation activity in the infected cells, suggesting that CPAFcp is necessary for the degradation of host transcription factors required for MHC antigen expression duringC. pneumoniaeinfection. These observations have revealed a unique molecular mechanism forC. pneumoniaeto evade host adaptive immunity that may aid in its persistence.
The Group B Streptococcal C5a Peptidase Is Both a Specific Protease and an Invasin
Authors: Cheng Qi , Stafslien Deborah , Purushothaman Sai Sudha , Cleary Patrick
Published: July 2002

The group B streptococcus (GBS) is a major cause of pneumonia, sepsis, and meningitis in neonates and a serious cause of mortality or morbidity in immunocompromised adults. Although these streptococci adhere efficiently and invade a variety of tissue-specific epithelial and endothelial cells, adhesins and invasins are still unknown. All serotypes of GBS studied to date express C5a peptidase (SCPB) on their surface. This investigation addresses the possibility that this relatively large surface protein has additional activities. Rabbit anti-SCPB serum inhibited invasion of lung epithelial A549 cells by the serotype Ia strain O90R, suggesting that SCPB is an invasin. This was confirmed by inserting an in-frame 25-amino-acid deletion into thescpBgene. Invasion of HEp2 and A549 human cell lines was significantly reduced by the mutation. Enzyme-linked immunosorbent assays were used to demonstrate that purified SCPB protein binds directly to HEp2 and A549 cells and also binds the extracellular matrix protein fibronectin. Binding was dose dependent and saturable. These results suggested that SCPB is one of several potential invasins essential for GBS colonization of damaged epithelium.
The LTR72 Mutant of Heat-Labile Enterotoxin ofEscherichia coliEnhances the Ability of Peptide Antigens To Elicit CD4+T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin
Authors: Beignon A.-S. , Briand J.-P. , Rappuoli R. , Muller S. , Partidos C. D.
Published: July 2002

Application of antigens with an adjuvant onto bare skin is a needle-free and pain-free immunization procedure that delivers antigens to the immunocompetent cells of the epidermis. We tested here the immunogenicity and adjuvanticity of two mutants of heat-labile enterotoxin (LT) ofEscherichia coli, LTK63 and LTR72. Both mutants were shown to be immunogenic, inducing serum and mucosal antibody responses. The application of LTK63 and LTR72 to bare skin induced significant protection against intraperitoneal challenge with a lethal dose of LT. In addition, both LT mutants enhanced the capacity of peptides TT:830-843 and HA:307-319 (representing T-helper epitopes from tetanus toxin and influenza virus hemagglutinin, respectively) to elicit antigen-specific CD4+T cells after coapplication onto bare skin. However, only mutant LTR72 was capable of stimulating the secretion of high levels of gamma interferon. These findings demonstrate that successful skin immunization protocols require the selection of the right adjuvant in order to induce the appropriate type of antigen-specific immune responses in a selective and reliable way. Moreover, the use of adjuvants such the LTK63 and LTR72 mutants, with no or low residual toxicity, holds a lot of promise for the future application of vaccines to the bare skin of humans.
H-NS-Dependent Regulation of Flagellar Synthesis Is Mediated by a LysR Family Protein
Authors: Ko Minsu , Park Chankyu
Published: July 2002

H-NS regulates the flagellar master operon (
flhDC
) and thus is necessary for flagellation of
Escherichia coli
. However, the molecular mechanism of its regulation has remained unknown. Genetic screening of a transposon insertion abolishing the H-NS effect revealed a previously unidentified gene, named
hdfR
, encoding a LysR family protein. Binding of purified HdfR to the
flhDC
promoter was demonstrated by a DNA mobility shift assay, indicating that HdfR is a transcriptional regulator for the flagellar master operon. Furthermore, the expression of the
hdfR
gene was shown to be negatively regulated by H-NS.
Novel Insertion Sequence Elements Associated with Genetic Heterogeneity and Phenotype Conversion in
Ralstonia solanacearum
Authors: Jeong Eun-Lee , Timmis Jeremy N.
Published: July 2002

Three insertion sequences (IS) elements were isolated from the phytopathogen
Ralstonia solanacearum
. Southern hybridization using these IS elements as probes revealed hybridization profiles that varied greatly between different strains of the pathogen. During a spontaneous phenotype conversion event, the promoter of the
phcA
gene was interrupted by one of these IS elements.
Two Novel Genes Induced by Hard-Surface Contact of
Colletotrichum gloeosporioides
Conidia
Authors: Kim Yeon-Ki , Liu Zhi-Mei , Li Daoxin , Kolattukudy Pappachan E.
Published: July 2002

Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen,
Colletotrichum gloeosporioides
, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode
Colletotrichum
hard-surface induced proteins are designated
chip
genes. In this study, we report the characterization of
CHIP2
and
CHIP3
genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. The
CHIP2
product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The
CHIP3
product would be a nine-transmembrane-domain-containing protein. RNA blots showed that
CHIP2
and
CHIP3
are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting that
C. gloeosporioides
might have genes functionally redundant to
CHIP2
and
CHIP3
or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.
ς
70
Is the Principal Sigma Factor Responsible for Transcription of
acs
, Which Encodes Acetyl Coenzyme A Synthetase in
Escherichia coli
Authors: Kumari Suman , Simel Erica J. , Wolfe Alan J.
Published: July 2002

Cells of
Escherichia coli
undergo a metabolic switch associated with the production and utilization of acetate. During exponential growth on tryptone broth, these cells excrete acetate via the phosphotransacetylase-acetate kinase (Pta-AckA) pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. This metabolic switch depends upon the induction of Acs. As part of our effort to dissect the mechanism(s) underlying induction and to identify the signal(s) that triggers that induction, we sought the sigma factor most responsible for
acs
expression. Using isogenic strains that carry a temperature sensitivity allele of the gene that encodes ς
70
and either a wild-type or null allele of the gene that encodes ς
S
, we determined by immunoblotting, reverse transcriptase PCR, and
acs
::
lacZ
transcriptional fusion analyses that ς
70
is the sigma factor primarily responsible for the
acs
transcription that cells induce during mid-exponential phase. In contrast, ς
S
partially inhibits that transcription as cells enter stationary phase.
RNA Polymerases from
Bacillus subtilis
and
Escherichia coli
Differ in Recognition of Regulatory Signals In Vitro
Authors: Artsimovitch Irina , Svetlov Vladimir , Anthony Larry , Burgess Richard R. , Landick Robert
Published: July 2002

Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species,
Escherichia coli
and
Bacillus subtilis
, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general
E. coli
elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the ς subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the β and β′ subunits and the downstream DNA duplex.
Distribution of the Mosaic Structured
murM
Genes among Natural Populations of
Streptococcus pneumoniae
Authors: Filipe Sergio R. , Severina Elena , Tomasz Alexander
Published: July 2002

The presence and sequence variation of the
murM
gene were studied in a large collection (814 strains) of genetically diverse
Streptococcus pneumoniae
isolates, which included 27 different serogroups and both penicillin-resistant (423 isolates, 67 pulsed-field gel electrophoretic [PFGE] types) and intermediately penicillin-resistant (165 isolates, 66 PFGE types) and penicillin-susceptible (226 isolates, 135 PFGE types) strains. Diversity of the
murM
sequences was tested by hybridization with mainly two kinds of probes: one derived from the amplification of the nucleotide sequence between nucleotides 201 and 624 in the penicillin-susceptible laboratory strain R36A (
murMA
probe) and a second probe that amplified the comparable, highly divergent sequence in the penicillin-resistant strain Pen6 (
murMB
probe). The great majority of the strains (761 of 814), including both penicillin-susceptible and penicillin-resistant isolates, reacted exclusively with the
murMA
probe. A smaller group of penicillin-resistant strains (48 of 814 isolates) reacted only with the
murMB
DNA probe, and an additional 5 isolates reacted with both probes. High-pressure liquid chromatography analysis of the peptidoglycan of strains hybridizing with
murMB
showed that they invariably contained an increased proportion of branched peptides. Complete sequencing of
murM
from a group of penicillin-resistant isolates allowed the identification of a number of different
murMB
alleles that differed in the length and exact position of the divergent (Pen6 type) sequences within the particular
murM
. The close similarity of these divergent sequences in the various
murM
alleles suggests a possible common heterologous origin.
WdCHS3
, a Gene That Encodes a Class III Chitin Synthase in
Wangiella
(
Exophiala
)
dermatitidis
, Is Expressed Differentially under Stress Conditions
Authors: Wang Zheng , Szaniszlo Paul J.
Published: July 2002

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of
W. dermatitidis
as a probe, we isolated the chitin synthase gene,
WdCHS3
, from this polymorphic melanized pathogen of humans. Northern blotting showed that
WdCHS3
was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5′ upstream sequence of
WdCHS3
provided evidence for a negative regulatory element at between −780 and −1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of
WdCHS3
in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of
WdCHS3
gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5′ upstream sequence.
Natural Genetic Competence in
Bacillus subtilis
Natto OK2
Authors: Ashikaga Sayaka , Nanamiya Hideaki , Ohashi Yoshiaki , Kawamura Fujio
Published: July 2002

We isolated a
Bacillus subtilis
natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a
comG-lacZ
fusion at the
amyE
locus. Although transcription of the late competence genes was not detected in the
B. subtilis
natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the
mecA
or the
clpC
mutation derived from
B. subtilis
168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for
B. subtilis
168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the
mecA
and
clpC
mutants of strain OK2 (ComK
OK2
) could activate the transcription of the whole set of late competence genes and suggested that ComK
OK2
was not activated in strain OK2 during competence development. We therefore sequenced the
comS
gene of OK2 and compared it with that of 168. The
comS
OK2
had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.
An
n
-Alkane-Responsive Promoter Element Found in the Gene Encoding the Peroxisomal Protein of
Candida tropicalis
Does Not Contain a C
6
Zinc Cluster DNA-Binding Motif
Authors: Kanai Tamotsu , Hara Akihiro , Kanayama Naoki , Ueda Mitsuyoshi , Tanaka Atsuo
Published: July 2002

When an asporogenic diploid yeast,
Candida tropicalis
, is cultivated on
n
-alkane, the expression of the genes encoding enzymes of the peroxisomal β-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to
n
-alkane (UAS
ALK
) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of
C. tropicalis
(
CT-T3A
). The 29-bp region (from −289 to −261) present upstream of the TATA sequence was sufficient to induce
n
-alkane-dependent expression of a reporter gene. Besides
n
-alkane, UAS
ALK
-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UAS
ALK
were constructed and tested for their UAS activity. It was clarified that the important nucleotides for UAS
ALK
activity were located within 10-bp region from −273 to −264 (5′-TCCTGCACAC-3′). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of
Saccharomyces cerevisiae
. Similar sequences to UAS
ALK
were also found on several peroxisomal enzyme-encoding genes of
C. tropicalis.
A Long T · A Tract in the
upp
Initially Transcribed Region Is Required for Regulation of
upp
Expression by UTP-Dependent Reiterative Transcription in
Escherichia coli
Authors: Cheng Yulin , Dylla Sara M. , Turnbough Charles L.
Published: July 2002

In
Escherichia coli
, pyrimidine-mediated regulation of
upp
expression occurs by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be elongated. The
upp
initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Initiation can occur at either the first or the second base in this sequence (designated G6 and A7, with numbering from the promoter −10 region). High intracellular UTP levels favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive UMP addition) within the 8-bp T · A tract in the initially transcribed region and are aborted. In contrast, low intracellular UTP levels favor initiation at position G6, which results in transcripts that can, in part, avoid reiterative transcription and be elongated normally. In this study, we examined the regulatory requirement for the long T · A tract in the
upp
initially transcribed region. We constructed
upp
promoter mutations that shorten the T · A tract to 7, 6, 5, 4, 3, or 2 bp and examined the effects of these mutations on
upp
expression and regulation. The results indicate that pyrimidine-mediated regulation is gradually reduced as the T · A tract is shortened from 7 to 3 bp; at which point regulation ceases. This reduction in regulation is due to large-percentage increases in
upp
expression in cells grown under conditions of pyrimidine excess. Quantitation of cellular transcripts and in vitro transcription studies indicate that the observed effects of a shortened T · A tract on
upp
expression and regulation are due to increases in the fraction of both G6- and A7-initiated transcripts that avoid reiterative transcription and are elongated normally.
Evidence against an Interaction between the mRNA Downstream Box and 16S rRNA in Translation Initiation
Authors: Moll Isabella , Huber Michael , Grill Sonja , Sairafi Pooneh , Mueller Florian , Brimacombe Richard , Londei Paola , Bläsi Udo
Published: July 2002

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless λ
c
I-
lacZ
reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973–8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393–1402, 2000), we conclude that the db does not base pair with the adb.
Cs
+
Induces the
kdp
Operon of
Escherichia coli
by Lowering the Intracellular K
+
Concentration
Authors: Jung Kirsten , Krabusch Mechthild , Altendorf Karlheinz
Published: July 2002

Cs
+
was found to induce expression of the
kdpFABC
operon, encoding a high-affinity K
+
uptake system of
Escherichia coli.
Quantitative expression analyses at the transcriptional and translational levels reveal that CsCl causes much higher induction of
kdpFABC
than does NaCl. A decrease of the intracellular K
+
concentration is found in cells exposed to CsCl. The results indicate that
kdpFABC
expression is induced when the intracellular K
+
concentration is lowered. Moreover, the results imply that the signal transduction cascade mediated by KdpD and KdpE is able to integrate multiple signals.
Control of the Arabinose Regulon in
Bacillus subtilis
by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping
Authors: Mota Luı́s Jaime , Sarmento Leonor Morais , de Sá-Nogueira Isabel
Published: July 2002

The proteins involved in the utilization of
l
-arabinose by
Bacillus subtilis
are encoded by the
araABDLMNPQ-abfA
metabolic operon and by the
araE
/
araR
divergent unit. Transcription from the
ara
operon,
araE
transport gene, and
araR
regulatory gene is induced by
l
-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the
araABDLMNPQ-abfA
(OR
A1
and OR
A2
) and
araE
(OR
E1
and OR
E2
) promoters and noncooperatively to a single operator in the
araR
(OR
R3
) promoter region. Here, we have investigated how AraR controls transcription from the
ara
regulon in vivo. A deletion analysis of the
ara
promoters region showed that the five AraR binding sites are the key
cis
-acting regulatory elements of their corresponding genes. Furthermore, OR
E1
-OR
E2
and OR
R3
are auxiliary operators for the autoregulation of
araR
and the repression of
araE
, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the
ara
operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the
araABDLMNPQ-abfA
operon and
araE
promoters, strongly suggesting that it is the major protein involved in the repression mechanism of
l
-arabinose-inducible expression in vivo. The
ara
regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.
Viability of
rep recA
Mutants Depends on Their Capacity To Cope with Spontaneous Oxidative Damage and on the DnaK Chaperone Protein
Authors: Bredèche Marie-Florence , Ehrlich S. Dusko , Michel Bénédicte
Published: July 2002

Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins. Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth.
rep recA
mutants are viable and display no growth defect at 37 or 42°C. The putative role of chaperone proteins in
rep
and
rep recA
mutants was investigated by testing the effects of
dnaK
mutations.
dnaK756
and
dnaK306
mutations, which allow growth of otherwise wild-type
Escherichia coli
cells at 40°C, are lethal in
rep recA
mutants at this temperature. Furthermore, they affect the growth of
rep
mutants, and to a lesser extent, that of
recA
mutants. We conclude that both
rep
and
recA
mutants require DnaK for optimal growth, leading to low viability of the triple (
rep recA dnaK
) mutant.
rep recA
mutant cells form colonies at low efficiency when grown to exponential phase at 30°C. Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30°C. The plating defect of
rep recA
mutant cells is suppressed by the presence of catalase in the plates. The cryosensitivity of
rep recA
mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.
Structure, Expression, and Functional Analysis of the Gene Coding for Calmodulin in the Chytridiomycete
Blastocladiella emersonii
Authors: Simão Rita de Cássia Garcia , Gomes Suely Lopes
Published: July 2002

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete
Blastocladiella emersonii
were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM.
B. emersonii
CaM has been expressed in
Escherichia coli
as a fusion protein with gluthatione
S
-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca
2+
,
B. emersonii
CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in
B. emersonii
, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit
B. emersonii
sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca
2+
channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca
2+
-CaM complex and CaMKII play an important role during growth and sporulation in
B. emersonii
.
The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane
Authors: Rydman Pia S. , Bamford Dennis H.
Published: July 2002

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble β-1,4-
N
-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.
Depletion of Free 30S Ribosomal Subunits in
Escherichia coli
by Expression of RNA Containing Shine-Dalgarno-Like Sequences
Authors: Mawn Mary V. , Fournier Maurille J. , Tirrell David A. , Mason Thomas L.
Published: July 2002

We have constructed synthetic coding sequences for the expression of poly(α,
l
-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in
Escherichia coli
. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5′-GAGGAGG-3′) that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.
Characterization of GlnK
1
from
Methanosarcina mazei
Strain Gö1: Complementation of an
Escherichia coli glnK
Mutant Strain by GlnK
1
Authors: Ehlers Claudia , Grabbe Roman , Veit Katharina , Schmitz Ruth A.
Published: July 2002

Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon
Methanosarcina mazei
strain Gö1 and show that
M. mazei
GlnK
1
is able to functionally complement an
Escherichia coli glnK
mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of
E. coli
GlnK.
M. mazei
GlnK
1
is encoded in the

glnK
1

-

amtB
1

operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal

glnK
1

shows 44% identity to the
E. coli
GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure.

M. mazei glnK
1

was cloned and overexpressed in
E. coli
, and GlnK
1
was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK
1
-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK
1
indicated significant differences compared to
E. coli
GlnK. First, GlnK
1
from
M. mazei
is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between
M. mazei
GlnK
1
and
Klebsiella pneumoniae
GlnK are not formed. Because
M. mazei
GlnK
1
was able to complement growth of an
E. coli glnK
mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.