bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast
Authors: Rodriguez-Vargas Sonia , Estruch Francisco , Randez-Gil Francisca
Published: July 2002

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for
TPI1
, which encodes triose-phosphate isomerase;
ERG10
, the gene for acetoacetyl coenzyme A thiolase; and
IMH1
, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of
TPI1
and
ERG10
genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of
ERG10
overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high
ERG10
expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an
erg10
mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in
Saccharomyces cerevisiae
and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.
Hepatitis E Virus Immunoglobulin G Antibodies in Different Populations in Campinas, Brazil
Authors: Gonçales Neiva S. L. , Pinho João Renato R. , Moreira Regina C. , Saraceni Cláudia P. , Spina Ângela M. M. , Stucchi Raquel B. , Filho Aírton D. Ribeiro , Magna Luís A. , Gonçales Júnior Fernando L.
Published: July 2002

The seroprevalence of anti-hepatitis E virus (HEV) antibodies was investigated by enzyme immunoassay in 205 volunteer blood donors, 214 women who attended a center for anonymous testing for human immunodeficiency virus (HIV) infection, and 170 hospital employees in Campinas, a city in southeastern Brazil. The prevalence of anti-HEV antibodies ranged from 2.6% (3 of 117) in health care professionals to 17.7% (38 of 214) in women who considered themselves at risk for HIV. The prevalence of anti-HEV antibodies in health care professionals was not significantly different from that in healthy blood donors (3.0%, 5 of 165) and blood donors with raised alanine aminotransferase levels (7.5%, 3 of 40). The prevalence of anti-HEV antibodies (13.2%, 7 of 53) in cleaning service workers at a University hospital was similar to that among women at risk for HIV infection. These results suggest that HEV is circulating in southeastern Brazil and that low socioeconomic status is an important risk factor for HEV infection in this region.
Feed a Cold, Starve a Fever?
Authors: van den Brink Gijs R. , van den Boogaardt Daniëlle E. M. , van Deventer Sander J. H. , Peppelenbosch Maikel P.
Published: July 2002

An English old wives’ tale advises us to “feed a cold and starve a fever.” Here we report that the nutritional status modulates the T helper 1 (Th1)-Th2 balance of activated T cells in human volunteers. Food intake resulted in increased levels of gamma interferon production, whereas food deprivation stimulated interleukin-4 release.
Comparison of Murex Single-Use Diagnostic System with Traditional Enzyme Immunoassay for Detection of Exposure to Human Immunodeficiency Virus
Authors: Martin Christin A. , Keren David F.
Published: July 2002

Because a retrospective study detected 13 negative Western blots out of 38 single-use diagnostic system (SUDS)-positive cases over a 1-year period, we performed a prospective study to compare the performance of the SUDS test with that of enzyme immunoassay (EIA). Of 888 SUDS-tested sera, 875 (98.4%) were both SUDS and EIA negative and 5 (0.6%) were SUDS, EIA, and Western blot positive. The rate of SUDS-positive samples decreased from 3.16/month in the retrospective study to 1.33/month in the prospective study. The immunoassays had sensitivities and specificities of 100 and 99.7 (SUDS) and 100 and 99.4% (traditional EIA), respectively. In laboratories with experienced personnel, the SUDS test performs as well as the EIA as a screen for infection with the human immunodeficiency virus.
CD4+T-Cell Subsets That Mediate Immunological Memory toMycobacterium tuberculosisInfection in Mice
Authors: Andersen Peter , Smedegaard Birgitte
Published: July 2002

We have studied CD4+T cells that mediate immunological memory to an intravenous infection withMycobacterium tuberculosis. The studies were conducted with a mouse model of memory immunity in which mice are rendered immune by a primary infection followed by antibiotic treatment and rest. Shortly after reinfection, tuberculosis-specific memory cells were recruited from the recirculating pool, leading to rapidly increasing precursor frequencies in the liver and a simultaneous decrease in the blood. A small subset of the infiltrating T cells was rapidly activated (<20 h) and expressed high levels of intracellular gamma interferon and the T-cell activation markers CD69 and CD25. These memory effector T cells expressed intermediate levels of CD45RB and were heterogeneous with regard to the L-selectin and CD44 markers. By adoptive transfer into nude mice, the highest level of resistance to a challenge withM. tuberculosiswas mediated by CD45RBhigh,l-selectinhigh, CD44lowcells. Taken together, these two lines of evidence support an important role for memory cells which have reverted to a naive phenotype in the long-term protection againstM. tuberculosis.
Two Allelic Forms of the Aureolysin Gene (
aur
) within
Staphylococcus aureus
Authors: Sabat Artur , Kosowska Klaudia , Poulsen Knud , Kasprowicz Andrzej , Sekowska Agnieszka , van den Burg Bertus , Travis James , Potempa Jan
Published: July 2002

Proteinases of
Staphylococcus aureus
are emerging as potential virulence factors which may be involved in the pathogenecity of staphylococcal diseases. We describe here the structure of the gene encoding the metalloproteinase referred to as aureolysin. This gene occurs in two allelic forms and is strongly conserved among
S. aureus
strains, implying the possibility that the proteinase may have important housekeeping functions.
Immunization with Recombinant
Helicobacter pylori
Urease in Specific-Pathogen-Free Rhesus Monkeys (
Macaca mulatta
)
Authors: Solnick Jay V. , Canfield Don R. , Hansen Lori M. , Torabian Sima Z.
Published: July 2002

Immunization with urease can protect mice from challenge with
Helicobacter pylori
, though results vary depending on the particular vaccine, challenge strain, and method of evaluation. Unlike mice, rhesus monkeys are naturally colonized with
H. pylori
and so may provide a better estimate of vaccine efficacy in humans. The purpose of this study was to examine the effectiveness of
H. pylori
urease as a vaccine in specific-pathogen (
H. pylori
)-free rhesus monkeys. Monkeys raised from birth and documented to be free of
H. pylori
were vaccinated with orogastric (
n
= 4) or intramuscular (
n
= 5) urease. Two control monkeys were sham vaccinated. All monkeys were challenged with a rhesus monkey-derived strain of
H. pylori
, and the effects of vaccination were evaluated by use of quantitative cultures of gastric tissue, histology, and measurement of serum immunoglobulin G (IgG) and salivary IgA. Despite a humoral immune response, all monkeys were infected after
H. pylori
challenge, and there were no differences in the density of colonization. Immunization with urease therefore does not fully protect against challenge with
H. pylori
. An effective vaccine to prevent
H. pylori
infection will require different or more likely additional antigens, as well as improvements in the stimulation of the host immune response.
Major Carbohydrate Antigen ofEchinococcus multilocularisInduces an Immunoglobulin G Response Independent of αβ+CD4+T Cells
Authors: Dai Wen Juan , Hemphill Andrew , Waldvogel Andreas , Ingold Katrin , Deplazes Peter , Mossmann Horst , Gottstein Bruno
Published: July 2002

Echinococcus multiloculariscauses alveolar echinococcosis, one of the most lethal helminthic (accidental) infections in humans, as the life cycle predominantly includes wildlife rodents as intermediate hosts. The physical barrier between the proliferating parasitic metacestode and the host tissue is the acellular laminated layer (LL), which is characterized by its rich high-molecular-weight polysaccharide composition. Conversely to a crude protein-rich vesicular fluid antigen, a major carbohydrate antigen of the LL—the Em2(G11) antigen—did not stimulate murine T-cell proliferation in vitro. In fact, the persistent metacestode growth and antigenic stimulation induced a Th2 shift in vivo following conventional infection by intraperitoneal inoculation of 100 metacestode vesicles into C57/BL6 mice. Concurrently, the expression of Th1 cytokines (interleukin-2 and gamma interferon) remained persistently low until the late stage of chronic infection. In comparison to a recombinant proteinic II/3 antigen, the specific immunoglobulin G (IgG) response against the Em2(G11) antigen (including all IgG isotypes) maintained persistently low avidity. Furthermore, the Em2(G11) antigen induced a specific IgM and IgG response in T-cell-deficient athymic nude, TCRβ−/−, major histocompatibility complex class II (MHCII)−/−(CD4-deficient), and CD40−/−mice. The Em2(G11)-specific IgG synthesized in nude TCRβ−/−and MHCII−/−mice was predominantly of the IgG3 and IgG2a isotypes and of the IgG3 and IgG2b isotypes in CD40−/−mice. This finding suggested that in vivo, the IgG response to major carbohydrate antigen Em2(G11) ofE. multiloculariscould take place independently of αβ+CD4+T cells and in the absence of CD40-CD40 ligand interactions; thus, the Em2(G11) antigen of the acellular LL represents a T-cell-independent antigen. Functionally, the encapsulating LL, and especially its major carbohydrate antigen, Em2(G11), seems to be one of the key factors in the parasite's survival strategy and acts by modulating the host immune response by virtue of its T-cell-independent nature.
Identification of a Locus Involved in Systemic Dissemination of
Yersinia enterocolitica
Authors: Nelson Kristin M. , Young Glenn M. , Miller Virginia L.
Published: July 2002

A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by
Yersinia enterocolitica
(G. M. Young and V. L. Miller, Mol. Microbiol. 25:319–328, 1997). An insertion in
hre20
, now designated
rscR
, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated in
rscR
, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in
rscR
was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal
rscR
gene and carrying
rscR
on a plasmid under the control of the inducible
araBAD
promoter was mutagenized with an mTn
5
Km-2 transposon containing a promoterless
lacZY
. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of
Serratia marcescens
, and a locus (designated
rscBAC
) with similarity to the
hmwABC
locus of
Haemophilus influenzae
. The
hmwABC
locus promotes adherence of
H. influenzae
to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993). A strain containing a deletion mutant of
rscA
, the
hmwA
homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the
rscR
mutant. This suggests that the phenotype of an
rscR
mutant is due to the loss of RscA.
CD8+-T-Cell Depletion Ameliorates Circulatory Shock inPlasmodium berghei-Infected Mice
Authors: Chang Wun-Ling , Jones Steven P. , Lefer David J. , Welbourne Tomas , Sun Guang , Yin Lijia , Suzuki Hodaka , Huang Jian , Granger D. Neil , van der Heyde Henri C.
Published: July 2002

ThePlasmodium berghei-infected mouse model is a well-recognized model for human cerebral malaria. Mice infected withP. bergheiexhibit (i) metabolic acidosis (pH < 7.3) associated with elevated plasma lactate concentrations, (ii) significant (P< 0.05) vascular leakage in their lungs, hearts, kidneys, and brains, (ii) significantly (P< 0.05) higher cell and serum glutamate concentrations, and (iv) significantly (P< 0.05) lower mean arterial blood pressures. Because these complications are similar to those of septic shock, the simplest interpretation of these findings is that the mice develop shock brought on by theP. bergheiinfection. To determine whether the immune system and specifically CD8+T cells mediate the key features of shock duringP. bergheimalaria, we depleted CD8+T cells by monoclonal antibody (mAb) treatment and assessed the complications of malarial shock.P. berghei-infected mice depleted of CD8+T cells by mAb treatment had significantly reduced vascular leakage in their hearts, brains, lungs, and kidneys compared with infected controls treated with rat immunoglobulin G. CD8-depleted mice were significantly (P< 0.05) protected from lactic acidosis, glutamate buildup, and diminished HCO3−levels. Although the blood pressure decreased in anti-CD8 mAb-treated mice infected withP. berghei, the cardiac output, as assessed by echocardiography, was similar to that of uninfected control mice. Collectively, our results indicate that (i) pathogenesis similar to septic shock occurs during experimentalP. bergheimalaria, (ii) respiratory distress with lactic acidosis occurs duringP. bergheimalaria, and (iii) most components of circulatory shock are ameliorated by depletion of CD8+T cells.
Inflammation and Clearance ofChlamydia trachomatisin Enteric and Nonenteric Mucosae
Authors: Igietseme Joseph U. , Portis John L. , Perry Linda L.
Published: July 2002

Immunization(s) fostering the induction of genital mucosa-targeted immune effectors is the goal of vaccines against sexually transmitted diseases. However, it is uncertain whether vaccine administration should be based on the current assumptions about the common mucosal immune system. We investigated the relationship between mucosal sites of infection, infection-induced inflammation, and immune-mediated bacterial clearance in mice using the epitheliotropic pathogenChlamydia trachomatis. Chlamydial infection of the conjunctival, pulmonary, or genital mucosae stimulated significant changes in tissue architecture with dramatic up-regulation of the vascular addressin, VCAM, a vigorous mixed-cell inflammatory response with an influx of α4β1+T cells, and clearance of bacteria within 30 days. Conversely, intestinal mucosa infection was physiologically inapparent, with no change in expression of the local MAdCAM addressin, no VCAM induction, no histologically detectable inflammation, and no tissue pathology. Microbial clearance was complete within 60 days in the small intestine but bacterial titers remained at high levels for at least 8 months in the large intestine. These findings are compatible with the notion that VCAM plays a functional role in recruiting cells to inflammatory foci, and its absence from the intestinal mucosa contributes to immunologic homeostasis at that site. Also, expression of type 1 T cell-mediated immunity to intracellularChlamydiamay exhibit tissue-specific variation, with the rate and possibly the mechanism(s) of clearance differing between enteric and nonenteric mucosae. The implications of these data for the common mucosal immune system and the delivery of vaccines against mucosal pathogens are discussed.
Gender Is a Major Factor in Determining the Severity of Mycoplasma Respiratory Disease in Mice
Authors: Yancey Anthony L. , Watson Harold L. , Cartner Sam C. , Simecka Jerry W.
Published: July 2002

Gender is a significant factor in determining the susceptibility to and severity of pulmonary diseases in both humans and animals. Murine respiratory mycoplasmosis (MRM), due to
Mycoplasma pulmonis
infection, is an excellent animal model for evaluation of the role of various host factors on the development of acute or chronic inflammatory lung diseases. MRM has many similarities to mycoplasma respiratory disease in humans. The purpose of the present study was to determine whether gender has a significant impact on lung disease due to
M. pulmonis
infection in mice. It was demonstrated that male mice consistently developed more severe disease in the lung parenchyma than did female mice. There was no gender difference in disease severity along the airways or any difference in mycoplasma numbers in lungs of male and female mice. Furthermore, surgical removal of reproductive organs reduced the severity of mycoplasma disease and the numbers of mycoplasma organisms recovered from lungs. Thus, gender plays a significant role in determining the severity of
M. pulmonis
disease. In fact, the gender of the host was a major factor in determining whether an acute or chronic inflammatory lung disease developed after infection with
M. pulmonis
.
Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode
Trichinella spiralis
Authors: Gounaris Kleoniki , Thomas Simon , Najarro Pilar , Selkirk Murray E.
Published: July 2002

The molecular components involved in the survival of the parasitic nematode
Trichinella spiralis
in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts γ-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.
Analysis of Mechanisms Associated with Loss of Infectivity of Clonal Populations of
Borrelia burgdorferi
B31MI
Authors: McDowell John V. , Sung Shian Ying , Labandeira-Rey Maria , Skare Jon T. , Marconi Richard T.
Published: July 2002

Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of
Borrelia burgdorferi
B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.
Improved Pattern for Genome-Based Screening Identifies Novel Cell Wall-Attached Proteins in Gram-Positive Bacteria
Authors: Janulczyk Robert , Rasmussen Magnus
Published: July 2002

With a large number of sequenced microbial genomes available, tools for identifying groups or classes of proteins have become increasingly important. Here we present an improved pattern for the identification of cell wall-attached proteins (CWPs), a group of proteins with diverse and important functions in gram-positive bacteria. This tripartite pattern is based on analysis of 65 previously described cell wall-attached proteins and takes into account the three principal requirements for cell wall sorting; a sortase target region (LPXTGX), a membrane-spanning region, and a charged stop-transfer tail. In five different genomes of gram-positive bacteria, the tripartite pattern identified a total of 35 putative CWPs, 19 of which were novel. The specificity and sensitivity of the tripartite pattern are higher than those of the classical pattern, which is based solely on the sortase target region. Several putative CWPs with atypical sortase target regions were identified. In the complete genome of the important human pathogenStreptococcus pyogenes, the tripartite pattern identified 14 putative CWPs. Seven of the putativeS. pyogenesproteins were novel, and two of these were a 5′ nucleotidase and a pullulanase. This study represents the first whole-genome screening for CWPs, and we conclude that the tripartite pattern is highly suitable for this purpose. Identification of CWPs using this pattern offers important possibilities in the study of the pathogenesis and physiology of gram-positive bacteria.
Concurrent Upregulation of Urokinase Plasminogen Activator Receptor and CD11b during Tuberculosis and Experimental Endotoxemia
Authors: Juffermans Nicole P. , Dekkers Pascale E. P. , Verbon Annelies , Speelman Peter , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

Patients with tuberculosis had higher expression of monocyte urokinase receptor (uPAR) and CD11b than controls. In vitro, lipoarabinomannan and lipopolysaccharide (LPS) fromEscherichia colishared the ability to enhance uPAR and CD11b expression on monocytes and granulocytes. In healthy volunteers, LPS induced increases in monocyte and granulocyte uPAR and CD11b.
Intracellular Survival of
Neisseria gonorrhoeae
in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A
Authors: Post Deborah M. B. , Phillips Nancy J. , Shao Jian Q. , Entz David D. , Gibson Bradford W. , Apicella Michael A.
Published: July 2002

Neisseria gonorrhoeae
is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of
N. gonorrhoeae
, the
msbB
gene was cloned and sequenced, a deletion and insertion mutation was introduced into
N. gonorrhoeae
, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the
msbB
LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the
msbB
mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the
N. gonorrhoeae msbB
mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec.
Effects ofAnaplasma phagocytophilaon NADPH Oxidase Components in Human Neutrophils and HL-60 Cells
Authors: Mott Jason , Rikihisa Yasuko , Tsunawaki Shohko
Published: July 2002

The human granulocytic ehrlichiosis agent,Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes.A. phagocytophilarapidly inhibits the superoxide anion (O2−) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence ofA. phagocytophilaon protein levels and localization of components of the NADPH oxidase were examined.A. phagocytophiladecreased levels of p22phox, but not gp91phox, p47phox, p67phox, or P40phoxreactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47phox, p67phox, Rac2, and p22phoxdid not colocalize withA. phagocytophilainclusions in neutrophils or HL-60 cells, and p22phoxlevels were also reduced.A. phagocytophiladid not prevent either membrane translocation of cytoplasmic p47phoxand p67phoxor phosphorylation of p47phoxupon stimulation by phorbol myristate acetate. The inhibitory signals for O2−generation was independent of several signals required forA. phagocytophilainternalization. These results suggest that rapid alteration in p22phoxinduced by binding ofA. phagocytophilato neutrophils is involved in the inhibition of O2−generation. Absence of colocalization of NADPH oxidase components with the inclusion further protectsA. phagocytophilafrom oxidative damage.
The Cytolethal Distending Toxin of
Haemophilus ducreyi
Inhibits Endothelial Cell Proliferation
Authors: Svensson Liselott A. , Henning Petra , Lagergård Teresa
Published: July 2002

Haemophilus ducreyi,
the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits mammalian cell proliferation. We investigated the effects of HdCDT on normal human endothelial cells and on tubule formation in an in vitro model of angiogenesis. Endothelial cells were arrested in the G
2
phase of the cell cycle, and tubule formation was inhibited in a dose-dependent manner. The antiproliferative activities of HdCDT on endothelial cells might contribute to the characteristic slow healing and persistence of chancroid ulcers.
Assessment of
flhDC
mRNA Levels in
Serratia liquefaciens
Swarm Cells
Authors: Tolker-Nielsen Tim , Christensen Allan Beck , Holmstrøm Kim , Eberl Leo , Rasmussen Thomas Bovbjerg , Sternberg Claus , Heydorn Arne , Molin Søren , Givskov Michael
Published: July 2002

We reported previously that artificial overexpression of the
flhDC
operon in liquid-grown
Serratia liquefaciens
resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, and M. Givskov, J. Bacteriol. 178:554–559, 1996). In the present report we show by means of reporter gene measurements, Northern analysis, and in situ reverse transcription-PCR that the amount of
flhDC
mRNA in surface-grown swarm cells does not exceed the maximum level found in nondifferentiated, vegetative cells. This suggests that surface-induced
S. liquefaciens
swarm cell differentiation, although dependent on
flhDC
gene expression, does not occur through elevated
flhDC
mRNA levels.
Deletion Analysis of the Flagellar Switch Protein FliG of
Salmonella
Authors: Kihara May , Miller Gabriele U. , Macnab Robert M.
Published: July 2002

The flagellar motor/switch complex, consisting of the three proteins FliG, FliM, and FliN, plays a central role in bacterial motility and chemotaxis. We have analyzed FliG, using 10-amino-acid deletions throughout the protein and testing the deletion clones for their motility and dominance properties and for interaction of the deletion proteins with the MS ring protein FliF. Only the N-terminal 46 amino acids of FliG (segments 1 to 4) were important for binding to FliF; consistent with this, an N-terminal fragment consisting of residues 1 to 108 bound FliF strongly, whereas a C-terminal fragment consisting of residues 109 to 331 did not bind FliF at all. Deletions in the region from residues 37 to 96 (segments 4 to 9), 297 to 306 (segment 30), and 317 to 326 (segment 32) permitted swarming, though not at wild-type levels; all other deletions caused paralyzed or, more commonly, nonflagellate phenotype. Except for those near the N terminus, deletions had a dominant negative effect on wild-type cells.
Mutations in
oxyR
Resulting in Peroxide Resistance in
Xanthomonas campestris
Authors: Mongkolsuk Skorn , Whangsuk Wirongrong , Fuangthong Mayuree , Loprasert Suvit
Published: July 2002

A spontaneous
Xanthomonas campestris
pv. phaseoli H
2
O
2
-resistant mutant emerged upon selection with 1 mM H
2
O
2
. In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H
2
O
2
resistance phenotype was abolished in
oxyR
-minus derivatives of the mutant, suggesting that elevated levels and mutations in
oxyR
were responsible for the phenotype. Nucleotide sequence analysis of the
oxyR
mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in
oxyR
affected expression of genes in the
oxyR
regulon. Expression of an
oxyR
-regulated gene,
ahpC
, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed
ahpC
expression. By contrast, expression of
oxyR5
from the
X. campestris
pv. phaseoli H
2
O
2
-resistant mutant and its derivative
oxyR5
G197D with a single-amino-acid change on expression vectors activated
ahpC
expression in the absence of inducer. The other single-amino-acid mutant derivative of
oxyR5
L301R had effects on
ahpC
expression similar to those of the wild-type
oxyR
. However, when the two single mutations were combined, as in
oxyR5
, these mutations had an additive effect on activation of
ahpC
expression.
The Chromosomal Location of the
Bacillus subtilis
Sporulation Gene
spoIIR
Is Important for Its Function
Authors: Khvorova Anastasia , Chary Vasant K. , Hilbert David W. , Piggot Patrick J.
Published: July 2002

Formation of the asymmetrically located septum during sporulation of
Bacillus subtilis
results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment. The rest of the chromosome is then translocated into the prespore from the mother cell. Transcription of
spoIIR
is initiated in the prespore by RNA polymerase containing ς
F
soon after the septum is formed. The SpoIIR protein is required for the activation of the transcription program directed by ς
E
in the mother cell. The
spoIIR
locus is located at 324°, near the origin of replication (0/360°). We show here that movement of
spoIIR
to 28° had little effect on sporulation. However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency. At 283° sporulation was reduced to less than 20% of the level obtained when
spoIIR
was at its natural location, and movement to 190° reduced sporulation to about 6% of that level. These positional effects were also seen in the transcription of a
spoIIR-lacZ
fusion. In contrast, movement of other
spo-lacZ
fusions from 28° to 190° had little effect on their expression. These results suggest that
spoIIR
is the subject of “positional regulation,” in the sense that the chromosomal position of
spoIIR
is important for its expression and function.
Structural Analysis of
Bacillus subtilis
Spore Peptidoglycan during Sporulation
Authors: Meador-Parton Jennifer , Popham David L.
Published: July 2002

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing
Bacillus subtilis
forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.
Two Conserved Glutamates in the Bacterial Nitric Oxide Reductase Are Essential for Activity but Not Assembly of the Enzyme
Authors: Butland Gareth , Spiro Stephen , Watmough Nicholas J. , Richardson David J.
Published: July 2002

The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe
B
–heme-Fe dinuclear catalytic center rather than a Cu
B
–heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in
Paracoccus denitrificans
NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the
cycA
promoter to drive aerobic expression of NOR in
P. denitrificans
and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome
bc
complex in
Escherichia coli
. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in
P. denitrificans
and
E. coli
. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.
Regulatory Functions of Serine-46-Phosphorylated HPr in
Lactococcus lactis
Authors: Monedero Vicente , Kuipers Oscar P. , Jamet Emmanuel , Deutscher Josef
Published: July 2002

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of
Lactococcus lactis
participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a
ptsH1
mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with
L. lactis
vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as
methyl
β-
d
-thiogalactoside (TMG) and 2-deoxy-
d
-glucose (2-DG). In vivo experiments with the
ptsH1
mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and
ptsH1
mutant strains, whereas TMG expulsion was slowed in the
ptsH1
mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.
Amplification of Mutator Cells in a Population as a Result of Horizontal Transfer
Authors: Funchain Pauline , Yeung Annie , Stewart Jean , Clendenin Wendy M. , Miller Jeffrey H.
Published: July 2002

Mutator cells that lack the mismatch repair system (MMR
−
) occur at rates of 10
−5
or less in laboratory populations started from wild-type cells. We show that after selection for recombinants in an interspecies mating between
Salmonella enterica
serovar Typhimurium and
Escherichia coli
, the percentage of MMR
−
cells rises to several percent of the recombinant population, and after a second successive mating and selection, greater than 95% of the recombinants are MMR
−
. Coupling a single cross and selection with either mutagenesis or selection for spontaneous mutants also results in a dramatic increase in MMR
−
cells. We discuss how horizontal transfer can result in mutator strains during adaptive evolution.
Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis
Authors: Jobling Michael G. , Holmes Randall K.
Published: July 2002

Cholera toxin (CT) is the prototype for theVibrio cholerae-Escherichia colifamily of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.
Eukaryotic Translation Initiation Factor 4E-Dependent Translation Is Not Essential for Survival of Starved Yeast Cells
Authors: Paz Irit , Choder Mordechai
Published: July 2002

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5′ cap structure (m
7
GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast
Saccharomyces cerevisiae CDC33
gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the
cdc33
temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved
cdc33
mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.
Differential Regulation of
ftsZ
Transcription during Septation of
Streptomyces griseus
Authors: Kwak Jangyul , Dharmatilake Amitha J. , Jiang Hao , Kendrick Kathleen E.
Published: July 2002

Streptomyces
has been known to form two types of septa. The data in this research demonstrated that
Streptomyces griseus
forms another type of septum near the base of sporogenic hyphae (basal septum). To understand the regulation of the septation machinery in
S. griseus
, we investigated the expression of the
ftsZ
gene. S1 nuclease protection assays revealed that four
ftsZ
transcripts were differentially expressed during morphological differentiation. The vegetative transcript (emanating from P
veg
) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation. Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form. Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely. Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5′ end point of the transcripts. The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays. The aseptate phenotype of the P
spo
mutant indicated that the increased transcription from P
spo
is required for sporulation septation, but not for vegetative or basal septum formation.
Initiation of Anaerobic Degradation of
p
-Cresol by Formation of 4-Hydroxybenzylsuccinate in
Desulfobacterium cetonicum
Authors: Müller Jochen A. , Galushko Alexander S. , Kappler Andreas , Schink Bernhard
Published: July 2002

The anaerobic bacterium
Desulfobacterium cetonicum
oxidized
p
-cresol completely to CO
2
with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of
p
-cresol is activated by addition to fumarate, analogous to anaerobic toluene,
m
-xylene, and
m
-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from
p
-cresol and fumarate was detected at an initial rate of 0.57 nmol min
−1
(mg of protein)
−1
. This activity was specific for extracts of
p
-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via β-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of
p
-cresol via methyl group oxidation by
p
-cresol-methylhydroxylase in this bacterium.
SmcR-Dependent Regulation of Adaptive Phenotypes in
Vibrio vulnificus
Authors: McDougald Diane , Rice Scott A. , Kjelleberg Staffan
Published: July 2002

Vibrio vulnificus
contains homologues of the
V. harveyi luxR
and
luxS
genes. A null mutation in
smcR
(
luxR
) resulted in a defect in starvation survival, inhibition of starvation-induced maintenance of culturability that occurs when
V. vulnificus
is starved prior to low-temperature incubation, and increased expression of stationary-phase phenotypes.
Analysis of a Complete Library of Putative Drug Transporter Genes in
Escherichia coli
Authors: Nishino Kunihiko , Yamaguchi Akihito
Published: July 2002

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In
Escherichia coli
, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in
E. coli
and investigated their drug resistance phenotypes using an
E. coli
drug-sensitive mutant as a host.
E. coli
cells transformed with a plasmid carrying one of 20 ORFs, i.e.,
fsr
,
mdfA
,
yceE
,
yceL
,
bcr
,
emrKY
,
emrAB
,
emrD
,
yidY
,
yjiO
,
ydhE
,
acrAB
,
cusA
(formerly
ybdE
),
yegMNO
,
acrD
,
acrEF
,
yhiUV
,
emrE
,
ydgFE
, and
ybjYZ
, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs,
cusA
,
yegMNO
,
ydgFE
,
yceE
,
yceL
,
yidY,
and
ybjYZ
are novel drug resistance genes. The
fsr, bcr, yjiO
,
ydhE, acrD,
and
yhiUV
genes gave broader resistance spectra than previously reported.
GerN, an Endospore Germination Protein of
Bacillus cereus
, Is an Na
+
/H
+
-K
+
Antiporter
Authors: Southworth Thomas W. , Guffanti Arthur A. , Moir Anne , Krulwich Terry A.
Published: July 2002

GerN, a
Bacillus cereus
spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na
+
-sensitive phenotype of an
Escherichia coli
mutant that is deficient in Na
+
/H
+
antiport activity (strain KNabc). GerN also reduced the concentration of K
+
required to support growth of an
E. coli
mutant deficient in K
+
uptake (strain TK2420). In a fluorescence-based assay of everted
E. coli
KNabc membrane vesicles, GerN exhibited robust Na
+
/H
+
antiport activity, with a

K
m

for Na
+
estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li
+
, but not K
+
, served as a substrate. GerN-mediated Na
+
/H
+
antiport was further demonstrated in everted vesicles as energy-dependent accumulation of
22
Na
+
. GerN also used K
+
as a coupling ion without completely replacing H
+
, as indicated by partial inhibition by K
+
of H
+
uptake into right-side-out vesicles loaded with Na
+
. K
+
translocation as part of the antiport was supported by the stimulatory effect of intravesicular K
+
on
22
Na
+
uptake by everted vesicles and the dependence of GerN-mediated
86
Rb
+
efflux on the presence of Na
+
in
trans
. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na
+
/H
+
-K
+
antiport. GerN-mediated Na
+
/H
+
-K
+
antiport was much more rapid than GerN-mediated Na
+
/H
+
antiport.