bluuebunny/crossref_metadata_2025 huggingface dataset:
VraA (BBI16) Protein of
Borrelia burgdorferi
Is a Surface-Exposed Antigen with a Repetitive Motif That Confers Partial Protection against Experimental Lyme Borreliosis
Authors: Labandeira-Rey Maria , Baker Elizabeth A. , Skare Jonathan T.
Published: July 2002

We have previously described the expression cloning of nine
Borrelia burgdorferi
antigens, using rabbit serum enriched for antibodies specific for infection-associated antigens, and determined that seven of these antigens were associated with infectious
B. burgdorferi
strain B31. One of these infection-associated antigens encoded a 451-amino-acid putative lipoprotein containing 21 consecutive and invariant 9-amino-acid repeat sequences near the amino terminus that we have designated VraA for virulent strain-associated repetitive antigen A. The
vraA
locus (designated BBI16 by The Institute for Genomic Research) maps to one of the 28-kb linear plasmids (designated lp28-4) that is not present in noninfectious strain B31 isolates. Subsequent PCR analysis of clonal isolates of
B. burgdorferi
B31 from infected mouse skin revealed a clone that lacked only lp28-4. Southern blot and Western blot analyses indicated that the lp28-4 and VraA proteins, respectively, were missing from this clone. We have also determined that VraA is a surface-exposed protein based on protease accessibility assays of intact whole cells. Furthermore,
vraA
expression is modestly derepressed when cells are grown at 37°C relative to cells grown at 32°C, suggesting that VraA is, in part, a temperature-inducible antigen. Homologues cross-reactive to
B. burgdorferi
B31 VraA, most with different molecular masses, were identified in several
B. burgdorferi
sensu lato isolates, including
B. andersonii
, suggesting that the immunogenic epitope(s) present in strain B31 VraA is conserved between
Borrelia
spp. In protection studies, only 8.3% of mice (1 of 12) immunized with full-length recombinant VraA fused to glutathione
S
-transferase (GST) were susceptible to infectious challenge with 10
2
B. burgdorferi
strain B31, whereas naive mice or mice immunized with GST alone were infected 40% or 63 to 67% (depending on tissues assayed) of the time, respectively. As such, the partial protection elicited by VraA immunization provides an additional testable vaccine candidate to help protect against Lyme borreliosis.
cDNA Array Analysis of Altered Gene Expression in Human Endothelial Cells in Response toChlamydia pneumoniaeInfection
Authors: Coombes Brian K. , Mahony James B.
Published: July 2002

Strong epidemiological and pathological evidence supports a role forChlamydia pneumoniaeinfection in atherosclerosis and human coronary heart disease. Animal models have shown thatC. pneumoniaedisseminates hematogenously in infected monocytes and macrophages, while in vitro data suggest that infected macrophages can transmitC. pneumoniaeinfection directly to endothelial cells. Endothelial cells may be key in vivo targets forC. pneumoniaeinfection; given that these cells are important in regulating the dynamics of the vessel wall, we used cDNA microarrays to study the transcriptional response of endothelial cells to infection withC. pneumoniae. cDNA arrays were used to characterize the mRNA expression profiles for 268 human genes following infection withC. pneumoniae, which were compared to mRNA profiles of uninfected cells. Selected genes of interest were further investigated by reverse transcription-PCR throughout a 24-h period of infection.C. pneumoniaeinfection upregulated mRNA expression for approximately 20 (8%) of the genes studied. Genes coding for cytokines (interleukin-1), chemokines (monocyte chemotactic protein 1 and interleukin-8), and cellular growth factors (heparin-binding epidermal-like growth factor, basic fibroblast growth factor, and platelet-derived growth factor B chain) were the most prominently upregulated. In addition to these families of genes, increases in mRNA levels for intracellular kinases and cell surface receptors with signal transduction activities were observed. Time course experiments showed that mRNA levels were upregulated within 2 h following infection. These results expand our knowledge of the response of endothelial cells toC. pneumoniaeby further defining the repertoire ofC. pneumoniae-inducible genes and provide new insight into potential mechanisms of atherogenesis. In addition, the use of cDNA microarrays may prove useful for the study of host cell responses toC. pneumoniaeinfection during latent and replicative stages of infection and related pathology.
Cellular Basis of Early Cytokine Response toPlasmodium falciparum
Authors: Hensmann Meike , Kwiatkowski Dominic
Published: July 2002

Uncertainty remains about the cellular origins of the earliest phase of the proinflammatory cytokine response to malaria. Here we show by fluorescence-activated cell sorter analysis that γδ T cells and CD14+cells from nonimmune donors produce tumor necrosis factor and that γδ T cells also produce gamma interferon within 18 h of contact with mycoplasma-freePlasmodium falciparum-infected erythrocytes in vitro. This early cytokine response is more effectively induced by intact than by lysed parasitized erythrocytes. However, the IFN-γ response to lysed parasites is considerably enhanced several days after peripheral blood mononuclear cells are primed with low numbers of intact parasitized erythrocytes, and in this case it derives from both αβ and γδ T cells. These data show that naı̈ve γδ T cells can respond very rapidly to malaria infection but that malaria fever may involve a multistage process in which the priming of both γδ and αβ T-cell populations boosts the cytokine response to lysed parasite products released at schizont rupture.
SarS, a SarA Homolog Repressible by
agr
, Is an Activator of Protein A Synthesis in
Staphylococcus aureus
Authors: Cheung Ambrose L. , Schmidt Katherine , Bateman Brian , Manna Adhar C.
Published: July 2002

The expression of protein A (
spa
) is repressed by global regulatory loci
sarA
and
agr
. Although SarA may directly bind to the
spa
promoter to downregulate
spa
expression, the mechanism by which
agr
represses
spa
expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the
spa
gene. The expression of
sarS
was almost undetectable in parental strain RN6390 but was highly expressed in
agr
and
sarA
mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a
sarS
mutant as detected in an immunoblot but returned to near-parental levels in a complemented
sarS
mutant. Transcriptional fusion studies with a 158- and a 491-bp
spa
promoter fragment linked to the
xylE
reporter gene disclosed that the transcription of the
spa
promoter was also downregulated in the
sarS
mutant compared with the parental strain. Interestingly, the enhancement in
spa
expression in an
agr
mutant returned to a near-parental level in the
agr sarS
double mutant but not in the
sarA sarS
double mutant. Correlating with this divergent finding is the observation that enhanced
sarS
expression in an
agr
mutant was repressed by the
sarA
locus supplied in
trans
but not in a
sarA
mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp
spa
promoter. Taken together, these data indicated that the
agr
locus probably mediates
spa
repression by suppressing the transcription of
sarS
, an activator of
spa
expression. However, the pathway by which the
sarA
locus downregulates
spa
expression is
sarS
independent.
Activation of Protein Tyrosine Kinases byCoxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis
Authors: Meconi Sonia , Capo Christian , Remacle-Bonnet Maryse , Pommier Gilbert , Raoult Didier , Mege Jean-Louis
Published: July 2002

Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) byC. burnetiiin THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflectsC. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation inC. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced byC. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, preventedC. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulentC. burnetiibut had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation byC. burnetiinegatively regulates bacterial uptake by interfering with cytoskeleton organization.
Oral Immunization with RecombinantStreptococcus gordoniiExpressingPorphyromonas gingivalisFimA Domains
Authors: Sharma Ashu , Honma Kiyonobu , Evans Richard T. , Hruby Dennis E. , Genco Robert J.
Published: July 2002

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis.P. gingivalisfimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation.P. gingivalisfimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development againstP. gingivalis-associated periodontitis. We have previously shown that biologically active domains ofP. gingivalisfimbrillin can be expressed on the surface of the human commensal bacteriumStreptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with twoS. gordoniirecombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes ofP. gingivalisFimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss followingP. gingivalisinfection was also evaluated. The results of this study show that the oral delivery ofP. gingivalisFimA epitopes viaS. gordoniivectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequentP. gingivalis-induced alveolar bone loss. These results support the potential usefulness of theS. gordoniivectors expressingP. gingivalisfimbrillin as a mucosal vaccine against adult periodontitis.
Coinfection withBorrelia burgdorferiand the Agent of Human Granulocytic Ehrlichiosis Alters Murine Immune Responses, Pathogen Burden, and Severity of Lyme Arthritis
Authors: Thomas Venetta , Anguita Juan , Barthold Stephen W. , Fikrig Erol
Published: July 2002

Lyme disease and human granulocytic ehrlichiosis (HGE) are tick-borne illnesses caused byBorrelia burgdorferiand the agent of HGE, respectively. We investigated the influence of dual infection withB. burgdorferiand the HGE agent on the course of murine Lyme arthritis and granulocytic ehrlichiosis. Coinfection resulted in increased levels of both pathogens and more severe Lyme arthritis compared with those in mice infected withB. burgdorferialone. The increase in bacterial burden during dual infection was associated with enhanced acquisition of both organisms by larval ticks that were allowed to engorge upon infected mice. Coinfection also resulted in diminished interleukin-12 (IL-12), gamma interferon (IFN-γ), and tumor necrosis factor alpha levels and elevated IL-6 levels in murine sera. During dual infection, IFN-γ receptor expression on macrophages was also reduced, implying a decrease in phagocyte activation. These results suggest that coinfection of mice withB. burgdorferiand the HGE agent modulates host immune responses, resulting in increased bacterial burden, Lyme arthritis, and pathogen transmission to the vector.
Helicobacter pyloriActivates the Cyclin D1 Gene through Mitogen- Activated Protein Kinase Pathway in Gastric Cancer Cells
Authors: Hirata Yoshihiro , Maeda Shin , Mitsuno Yuzo , Akanuma Masao , Yamaji Yutaka , Ogura Keiji , Yoshida Haruhiko , Shiratori Yasushi , Omata Masao
Published: July 2002

Helicobacter pyloriinduces cellular proliferation in host cells, but the mechanism remains unclear. Thus, we examined the effect ofH. pylorion cyclin D1, an important regulator of the cell cycle, especially in relation to intracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS) cells was enhanced by coculture withH. pyloristrain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form ofvacA also increased cyclin D1 transcription, but mutant forms ofcagEor the entirecagpathogenicity island did not enhance cyclin D1 transcription. These effects were confirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation byH. pyloriwas inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporter plasmid having any point mutations of the NF-κB binding sites in the promoter (pD1-κB1M, pD1-κB2M, or pD1-κB1/2M) or cotransfection of dominant negative IκBα did not affect cyclin D1 activation byH. pylori. In conclusion,H. pyloriactivates cyclin D1 through the mitogen-activated protein kinase pathway and not through NF-κB activation in AGS cells. This activation of cyclin D1 is partly dependent on thecagpathogenicity island but not onvacA.
Immune Response Induction and New Effector Mechanisms Possibly Involved in Protection Conferred by the Cuban Anti-Meningococcal BC Vaccine
Authors: Pérez Oliver , Lastre Miriam , Lapinet José , Bracho Gustavo , Dı́az Miriam , Zayas Caridad , Taboada Carlos , Sierra Gustavo
Published: July 2002

This report explores the participation of some afferent mechanisms in the immune response induced by the Cuban anti-meningococcal vaccine VA-MENGOC-BC. The induction of delayed-type hypersensitivity in nursing babies and lymphocyte proliferation after immunization is demonstrated. The presence of gamma interferon IFN-γ and interleukin-2 (IL-2) mRNAs but absence of IL-4, IL-5, and IL-10 mRNAs were observed in peripheral blood mononuclear cells from immunized subjects after in vitro challenge with outer membrane vesicles. In addition, some effector functions were also explored. The presence of opsonic activity was demonstrated in sera from vaccinees. The role of neutrophils as essential effector cells was shown. In conclusion, we have shown that, at least in the Cuban adult population, VA-MENGOC-BC induces mechanisms with a T-helper 1 pattern in the afferent and effector branches of the immune response.
Leishmania donovanip36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis
Authors: Melby Peter C. , Yang Jue , Zhao Weiguo , Perez Luis E. , Cheng Jun
Published: July 2002

The acquisition of immunity following subclinical or resolved infection with the intracellular parasiteLeishmania donovanisuggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmaniahomolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murineL. majorinfection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneousL. majorinfection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of theLeishmaniap36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other thanL. majorhas not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-γ but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemicL. donovanichallenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.
Gamma Interferon-Induced Inhibition ofToxoplasma gondiiin Astrocytes Is Mediated by IGTP
Authors: Halonen Sandra K. , Taylor Gregory A. , Weiss Louis M.
Published: July 2002

Toxoplasma gondiiis an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-γ) is the main cytokine preventing reactivation ofToxoplasmaencephalitis in the brain. Microglia are important IFN-γ-activated effector cells controlling the growth ofT. gondiiin the brain via a nitric oxide (NO)-mediated mechanism. IFN-γ can also activate astrocytes to inhibit the growth ofT. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasmamechanisms. In this study we investigated the role ofIGTP, a recently identified IFN-γ-regulated gene, in IFN-γ inhibition ofT. gondiiin murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-γ, and then tested for anti-Toxoplasmaactivity. In wild-type astrocytesT. gondiigrowth was significantly inhibited by IFN-γ, whereas in astrocytes from IGTP-deficient mice IFN-γ did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-γ induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-γ-induced inhibition ofT. gondiiin murine astrocytes.
Visualization of
Proteus mirabilis
within the Matrix of Urease-Induced Bladder Stones during Experimental Urinary Tract Infection
Authors: Li Xin , Zhao Hui , Lockatell C. Virginia , Drachenberg Cinthia B. , Johnson David E. , Mobley Harry L. T.
Published: July 2002

The virulence of a urease-negative mutant of uropathogenic
Proteus mirabilis
and its wild-type parent strain was assessed by using a CBA mouse model of catheterized urinary tract infection. Overall, catheterized mice were significantly more susceptible than uncatheterized mice to infection by wild-type
P. mirabilis
. At a high inoculum, the urease-negative mutant successfully colonized bladders of catheterized mice but did not cause urolithiasis and was still severely attenuated in its ability to ascend to kidneys. Using confocal laser scanning microscopy and scanning electron microscopy, we demonstrated the presence of
P. mirabilis
within the urease-induced stone matrix. Alizarin red S staining was used to detect calcium-containing deposits in bladder and kidney tissues of
P. mirabilis
-infected mice.
Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras
Authors: Jobling Michael G. , Holmes Randall K.
Published: July 2002

Variants of cholera toxin B subunit (CTB) were made by bisulfite- and oligonucleotide-directed mutagenesis of thectxBgene. Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC). Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM1in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies. Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB. RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 + 54, 91, and 95. Tyrosine-12 was identified as a novel residue important for GM1binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM1in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM1binding, based on the crystal structure of CTB and the oligosaccharide of GM1, were not appreciably altered in their ability to bind GM1in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC. Hybrid B genes made by fusingctxBand the relatedEscherichia coliheat-labile enterotoxineltBgenes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM1as well as wild-type CTB. This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs). Each MAb had a unique pattern of reactivity with the panel of CTB variants. Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope. The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers. Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.
Bicarbonate Ion Stimulates the Expression of Locus of Enterocyte Effacement-Encoded Genes in Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Abe Hiroyuki , Tatsuno Ichiro , Tobe Toru , Okutani Akiko , Sasakawa Chihiro
Published: July 2002

Enterohemorrhagic
Escherichia coli
(EHEC) strains adhere to the intestinal mucosa and produce an attaching and effacing (A/E) lesion. Most of the genes required to produce A/E lesions are thought to be encoded by the 36-kb pathogenicity island termed the locus for enterocyte effacement (LEE). Although the mechanisms underlying the bacterial adherence, including the genes involved, are still poorly understood, the preferential adherence phenotype of EHEC is thought to depend on the nature of the genes and/or the response of these genes to changes in environmental conditions. To explore the environmental factors affecting EHEC adherence, we used an O157:H7 strain and investigated the optimal growth conditions for its adherence to Caco-2 cells. We observed that EHEC grown in Dulbecco's modified Eagle's medium (DMEM) adhered more efficiently to Caco-2 cells than EHEC grown in Luria-Bertani (LB) broth. Among the components of DMEM, only NaHCO
3
was found to remarkably stimulate bacterial adherence. When bacteria were grown in LB broth containing NaHCO
3
, the production of intimin, Tir, EspA, and EspB was greatly enhanced compared with the production in LB broth. Indeed, the transcription of
ler
required for LEE-encoded gene expression was promoted in response to the concentration of NaHCO
3
in LB broth. Since the concentration of NaHCO
3
in the lower intestinal tract has been shown to be relatively high compared with that in the upper small intestine, our results may imply that NaHCO
3
is an important signaling factor for promoting colonization of EHEC in the lower intestinal tract in humans.
In Vivo Splicing and Functional Characterization of
Mycobacterium leprae
RecA
Authors: Frischkorn Klaus , Springer Burkhard , Böttger Erik C. , Davis Elaine O. , Colston M. Joseph , Sander Peter
Published: July 2002

The RecA proteins from
Mycobacterium tuberculosis
and
Mycobacterium leprae
contain inteins. In contrast to the
M. tuberculosis
RecA, the
M. leprae
RecA is not spliced in
Escherichia coli
. We demonstrate here that
M. leprae
RecA is functionally spliced in
Mycobacterium smegmatis
and produces resistance toward DNA-damaging agents and homologous recombination.
Septal Localization of the Membrane-Bound Division Proteins of
Bacillus subtilis
DivIB and DivIC Is Codependent Only at High Temperatures and Requires FtsZ
Authors: Katis V. L. , Wake R. G. , Harry E. J.
Published: July 2002

Using immunofluorescence microscopy, we have examined the dependency of localization among three
Bacillus subtilis
division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.
Marinomonas mediterranea
MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant
Authors: Solano Francisco , Lucas-Elío Patricia , Fernández Eva , Sanchez-Amat Antonio
Published: July 2002

Marinomonas mediterranea
is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to
M. mediterranea
by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn
10
derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability.
Sorting Out Bacterial Viability with Optical Tweezers
Authors: Ericsson M. , Hanstorp D. , Hagberg P. , Enger J. , Nyström T.
Published: July 2002

We have developed a method, using laser, optical tweezers and direct microscopic analysis of reproductive potential and membrane integrity, to assess single-cell viability in a stationary-phase
Escherichia coli
population. It is demonstrated here that a reduction in cell integrity, determined by using the fluorescent nucleic acid stain propidium iodide, correlated well with a reduction in cell proliferating potential during the stationary-phase period studied. Moreover, the same cells that exhibited reduced integrity were found to be the ones that failed to divide upon nutrient addition. A small but significant number of the intact cells (496 of 7,466 [6.6%]) failed to replicate. In other words, we did not find evidence for the existence of a large population of intact but nonculturable cells during the stationary-phase period studied but it is clear that reproductive ability can be lost prior to the loss of membrane integrity. In addition, about 1% of the stationary-phase cells were able to divide only once upon nutrient addition, and in a few cases, only one of the two cells produced by division was able to divide a second time, indicating that localized cell deterioration, inherited by only one of the daughters, may occur. The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary-phase-induced bacterial death and population heterogeneity is discussed.
Characterization of PrpC fromBacillus subtilis, a Member of the PPM Phosphatase Family
Authors: Obuchowski M. , Madec E. , Delattre D. , Boël G. , Iwanicki A. , Foulger D. , Séror S. J.
Published: July 2002

We cloned theyloOgene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate,p-nitrophenol phosphate, and endogenousBacillus subtilisproteins. TheprkCandprpCgenes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.
Activation of the Multiple Drug Resistance Gene
MDR1
in Fluconazole-Resistant, Clinical
Candida albicans
Strains Is Caused by Mutations in a
trans
-Regulatory Factor
Authors: Wirsching Stephanie , Michel Sonja , Köhler Gerwald , Morschhäuser Joachim
Published: July 2002

Resistance of
Candida albicans
against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant
C. albicans
isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the
MDR1
gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for
MDR1
activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable
MDR1
activation were analyzed. Sequence analysis of the
MDR1
regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of
trans
-regulatory factors, a
GFP
reporter gene was placed under the control of the
MDR1
promoter from the fluconazole-susceptible
C. albicans
strain CAI4, which does not express the
MDR1
gene in vitro. This
MDR1P-GFP
fusion was integrated into the genome of the clinical
C. albicans
isolates with the help of the dominant selection marker
MPA
R
developed for the transformation of
C. albicans
wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident
MDR1
promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the
GFP
gene. These results demonstrate that the
MDR1
promoter was activated by a
trans
-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive
MDR1
expression.
Degradation of Heme in Gram-Negative Bacteria: the Product of the
hemO
Gene of Neisseriae Is a Heme Oxygenase
Authors: Zhu Wenming , Wilks Angela , Stojiljkovic Igor
Published: July 2002

A full-length heme oxygenase gene from the gram-negative pathogen
Neisseria meningitidis
was cloned and expressed in
Escherichia coli
. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the
E. coli
cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen
Corynebacterium diphtheriae
and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXα and carbon monoxide as the final products. Homologs of the
hemO
gene were identified and characterized in six commensal
Neisseria
isolates,
Neisseria lactamica
,
Neisseria subflava
,
Neisseria flava
,
Neisseria polysacchareae
,
Neisseria kochii
, and
Neisseria cinerea
. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.
Mutations in
catB
, the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distal
ben
Genes and Contribute to a Complex Regulatory Circuit in
Acinetobacter
sp. Strain ADP1
Authors: Cosper Nathaniel J. , Collier Lauren S. , Clark Todd J. , Scott Robert A. , Neidle Ellen L.
Published: July 2002

Mutants of the bacterium
Acinetobacter
sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate and
cis
,
cis
-muconate to activate expression of the
benABCDE
operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by
cat
gene-encoded enzymes. In this report, four spontaneous mutants were found to carry
catB
mutations that enabled BenM-independent growth on benzoate.
catB
encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate,
cis
,
cis
-muconate, is enzymatically produced from catechol by the
catA
-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the
catB
mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomal
benA
::
lacZ
transcriptional fusion were constructed and used to investigate how
catB
mutations affect growth on benzoate. All of the
catB
mutations increased
cis
,
cis
-muconate-activated
ben
gene expression in strains lacking BenM. A model is presented in which the
catB
mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular
cis
,
cis
-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate
ben
gene transcription. CatM normally responds to
cis
,
cis
-muconate to activate
cat
gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown
catB
mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels of
cis
,
cis
-muconate and increasing
catA
expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.
Conversion of the
Vibrio fischeri
Transcriptional Activator, LuxR, to a Repressor
Authors: Egland Kristi A. , Greenberg E. P.
Published: July 2002

The
Vibrio fischeri
luminescence (
lux
) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the
lux
box at a position centered 42.5 bases upstream of the transcriptional start of the
lux
operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with ς
70
RNA polymerase it can activate transcription of the
lux
operon. Both the truncated LuxR and RNA polymerase are required for binding to
lux
regulatory DNA in vitro. We have constructed an artificial
lacZ
promoter with the
lux
box positioned between and partially overlapping the consensus −35 and −10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant
Escherichia coli
. Furthermore, multiple
lux
boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to
lux
boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native
lux
operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in
E. coli
. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the
lux
promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to
lux
box-containing DNA.
Nitrate Assimilation Genes of the Marine Diazotrophic, Filamentous Cyanobacterium
Trichodesmium
sp. Strain WH9601
Authors: Wang Qingfeng , Li Hong , Post Anton F.
Published: July 2002

A 4.0-kb DNA fragment of
Trichodesmium
sp. strain WH9601 contained gene sequences encoding the nitrate reduction enzymes,
nirA
and
narB
. A third gene positioned between
nirA
and
narB
encodes a putative membrane protein with similarity to the nitrate permeases of
Bacillus subtilis
(NasA) and
Emericella nidulans
(CrnA). The gene was shown to functionally complement a Δ
nasA
mutant of
B. subtilis
and was assigned the name
napA
(nitrate permease). NapA was involved in both nitrate and nitrite uptake by the complemented
B. subtilis
cells.
napA
is distinct from the
nrt
genes that encode the nitrate transporter of freshwater cyanobacteria.
The
Bacillus subtilis yabG
Gene Is Transcribed by SigK RNA Polymerase during Sporulation, and
yabG
Mutant Spores Have Altered Coat Protein Composition
Authors: Takamatsu Hiromu , Kodama Takeko , Imamura Atsuo , Asai Kei , Kobayashi Kazuo , Nakayama Tatsuo , Ogasawara Naotake , Watabe Kazuhito
Published: July 2002

The expression of six novel genes located in the region from
abrB
to
spoVC
of the
Bacillus subtilis
chromosome was analyzed, and one of the genes,
yabG
, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that
yabG
mRNA was first detected from 4 h after the cessation of logarithmic growth (
T
4
) in wild-type cells and in a
gerE36
(GerE
−
) mutant but not in
spoIIAC
(SigF
−
),
spoIIGAB
(SigE
−
),
spoIIIG
(SigG
−
), and
spoIVCB
(SigK
−
) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the
yabG
gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of
yabG
spores in
l
-alanine and in a mixture of
l
-asparagine,
d
-glucose,
d
-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from
yabG
spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that
yabG
encodes a sporulation-specific protein which is involved in coat protein composition in
B. subtilis
.
Kinetic Analysis of
Clostridium cellulolyticum
Carbohydrate Metabolism: Importance of Glucose 1-Phosphate and Glucose 6-Phosphate Branch Points for Distribution of Carbon Fluxes Inside and Outside Cells as Revealed by Steady-State Continuous Culture
Authors: Guedon Emmanuel , Desvaux Mickaël , Petitdemange Henri
Published: July 2002

During the growth of
Clostridium cellulolyticum
in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by
C. cellulolyticum
was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in
C. cellulolyticum
was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262–3269, 1999).
Bacillus subtilis
Locus Encoding a Killer Protein and Its Antidote
Authors: Adler Elliot , Barák Imrich , Stragier Patrick
Published: July 2002

We have isolated mutations that block sporulation after formation of the polar septum in
Bacillus subtilis
. These mutations were mapped to the two genes of a new locus,
spoIIS
. Inactivation of the second gene,
spoIISB
, decreases sporulation efficiency by 4 orders of magnitude. Inactivation of the first gene,
spoIISA
, has no effect on sporulation but it fully restores sporulation of a
spoIISB
null mutant, indicating that SpoIISB is required only to counteract the negative effect of SpoIISA on sporulation. An internal promoter ensures the synthesis of an excess of SpoIISB over SpoIISA during exponential growth and sporulation. In the absence of SpoIISB, the sporulating cells show lethal damage of their envelope shortly after asymmetric septation, a defect that can be corrected by synthesizing SpoIISB only in the mother cell. However, forced synthesis of SpoIISA in exponentially growing cells or in the forespore leads to the same type of morphological damage and to cell death. In both cases protection against the killing effect of SpoIISA can be provided by simultaneous synthesis of SpoIISB. The
spoIIS
locus is unique to
B. subtilis
, and since it is completely dispensable for sporulation its physiological role remains elusive.
The
Agrobacterium tumefaciens rnd
Homolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid
tra
Gene Expression
Authors: Luo Zhao-Qing , Farrand Stephen K.
Published: July 2002

Conjugal transfer of
Agrobacterium tumefaciens
Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer,
N
-(3-oxo-octanoyl)-
l
-homoserine lactone. We isolated four Tn5-induced mutants of
A. tumefaciens
C58 deficient in TraR-mediated activation of
tra
genes on pTiC58Δ
accR
. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn
5
was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by
rnd
of
Escherichia coli
, an RNase known to be involved in tRNA processing. The wild-type allele of the
rnd
homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of
cya2
, surround
A. tumefaciens rnd
, but none of these genes exerted a detectable effect on the expression of the
tra
reporter. In the mutant,
traR
was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of
tra
, but not the growth rate, was partially restored by increasing the copy number of
traR
or by disrupting
traM
, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in
rnd
also slightly reduced expression of two tested
vir
genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in
tra
gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.
Identification of a DNA Binding Region in GerE from
Bacillus subtilis
Authors: Crater Dinene L. , Moran Charles P.
Published: July 2002

Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE from
Bacillus subtilis
is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the
sigK
promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the
sigK
promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.
New Plasmid System To Select for
Saccharomyces cerevisiae
Purine-Cytosine Permease Affinity Mutants
Authors: Wagner Renaud , Straub Marie-Laure , Souciet Jean-Luc , Potier Serge , de Montigny Jacky
Published: July 2002

The
FCY2
gene of
Saccharomyces cerevisiae
encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine. A structure-function model for this PCP has been recently proposed. In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.