bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Cytokine Regulation by Cholera Toxin and Type II Heat-Labile Toxins Involves Differential Regulation of CD40 Ligand on CD4+T Cells
Authors: Martin Michael , Metzger Daniel J. , Michalek Suzanne M. , Connell Terry D. , Russell Michael W.
Published: July 2002

Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4+T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4+T cells. In a coculture system, CT-treated CD4+T cells induced significantly less TNF-α and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4+T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.
Vibrio cholerae tolC
Is Required for Bile Resistance and Colonization
Authors: Bina James E. , Mekalanos John J.
Published: July 2002

TolC and its homologues are outer membrane proteins that are essential for the transport of many molecules across the cell envelope. In this study we characterized the gene encoding
Vibrio cholerae
TolC.
V. cholerae tolC
mutants failed to secrete the RTX cytotoxin, were hypersensitive to antimicrobial agents, and were deficient in intestinal colonization.
Opsonized VirulentEdwardsiella tardaStrains Are Able To Adhere to and Survive and Replicate within Fish Phagocytes but Fail To Stimulate Reactive Oxygen Intermediates
Authors: Rao Putanae S. Srinivasa , Lim Tit Meng , Leung Ka Yin
Published: July 2002

Edwardsiella tardais responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans. Interactions ofE. tardawith blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes. Furthermore, only avirulentE. tardaelicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes. TnphoAtransposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived fromE. tardaPPD130/91. Five of these mutants induced more ROI production in phagocytes than the wild-type strain. Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs. Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing. The present study showed that there are differences in the interactions of virulent and avirulentE. tardaorganisms with fish phagocytes and PhoA+fusion mutants that could be used successfully to identify virulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused byE. tarda.
Erysipelothrix rhusiopathiae
YS-1 as a Live Vaccine Vehicle for Heterologous Protein Expression and Intranasal Immunization of Pigs
Authors: Shimoji Yoshihiro , Oishi Eiji , Kitajima Takashi , Muneta Yoshihiro , Shimizu Shinya , Mori Yasuyuki
Published: July 2002

We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of
Erysipelothrix rhusiopathiae
YS-1 and have examined the ability of a such recombinant
E. rhusiopathiae
strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of
Mycoplasma hyopneumoniae
strain E-1 was successfully translocated and expressed on the
E. rhusiopathiae
YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of
E. rhusiopathiae.
BALB/c mice subcutaneously immunized with the
E. rhusiopathiae
recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous
E. rhusiopathiae
Fujisawa-SmR strain, showing the efficacy of this heterologous-antigen expression system as a vaccine against
E. rhusiopathiae
infection. To determine whether protective immune responses are induced in target species, newborn, specific-pathogen-free piglets were immunized intranasally with a recombinant strain designated YS-19. The immunized piglets developed specific anti-SpaA.1 immunoglobulin G (IgG) antibodies in their serum and were protected from death by erysipelas, showing that mucosal vaccination of piglets with YS-19 induces systemic immune responses. Furthermore, YS-19-immunized piglets showed higher levels of P97-specific IgA antibodies in the respiratory tract than did YS-1-immunized piglets. Thus,
E. rhusiopathiae
YS-1 appears to be a promising vaccine vector for mucosal delivery that can induce local and systemic immune responses.
Role for Fimbriae and Lysine-Specific Cysteine Proteinase Gingipain K in Expression of Interleukin-8 and Monocyte Chemoattractant Protein inPorphyromonas gingivalis-Infected Endothelial Cells
Authors: Nassar Hamdy , Chou Hsin-Hua , Khlgatian Mary , Gibson Frank C. , Dyke T. E. Van , Genco Caroline Attardo
Published: July 2002

Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogenPorphyromonas gingivalisis capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions ofP. gingivaliswith endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences ofP. gingivalisinfection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition ofP. gingivalisfimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with liveP. gingivalisstrain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherentP. gingivalis fimAmutant DPG3 or whenP. gingivaliswas preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment ofP. gingivalis-infected HUVEC with cytochalsin D, which preventedP. gingivalisinvasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC withE. coliLPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with liveP. gingivalis. Analysis ofP. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment ofP. gingivaliswith protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production inP. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with aP. gingivalismutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with theP. gingivaliswild-type strain. These results indicate thatP. gingivaliscan temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.
Cytokine and Chemokine Production by Human Oral and Vaginal Epithelial Cells in Response toCandida albicans
Authors: Steele Chad , Fidel Paul L.
Published: July 2002

Oropharyngeal and vaginal candidiases are the most common forms of mucosal fungal infections and are primarily caused byCandida albicans, a dimorphic fungal commensal organism of the gastrointestinal and lower female reproductive tracts. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral and vaginal mucosa duringC. albicansinfections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and sinceC. albicansis closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines and/or chemokines produced by primary oral and vaginal epithelial cells and cell lines in response toC. albicans. The results showed that proinflammatory cytokines were produced by oral and/or vaginal epithelial cells at various levels constitutively with considerable interleukin-1α (IL-1α) and tumor necrosis factor alpha, but not IL-6, produced in response toC. albicans. In contrast, Th1-type (IL-12 and gamma interferon) and Th2-type-immunoregulatory (IL-10 and transforming growth factor β) cytokines and the chemokines monocyte chemoattractant protein 1 and IL-8 were produced in low to undetectable concentrations with little additional production in response toC. albicans. Taken together, these results indicate that cytokines and chemokines are variably produced by oral and vaginal epithelial cells constitutively, as well as in response toC. albicans, and are predominated by proinflammatory cytokines.
Identification by Mass Spectrometry of CD8+-T-CellMycobacterium tuberculosisEpitopes within the Rv0341 Gene Product
Authors: Flyer David C. , Ramakrishna Venkatesh , Miller Cara , Myers Helen , McDaniel Melanie , Root Karen , Flournoy Caroline , Engelhard Victor H. , Canaday David H. , Marto Jarrod A. , Ross Mark M. , Hunt Donald F. , Shabanowitz Jeffrey , White Forest M.
Published: July 2002

Identification ofMycobacterium tuberculosisproteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes fromM. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a singleM. tuberculosisprotein that is encoded by theM. tuberculosisRv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8+T cells from healthy blood donors in vitro and capable of recognizing and lysingM. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification ofM. tuberculosisproteins that can be evaluated as potential vaccine candidates.
Bacteria Induce Osteoclastogenesis via an Osteoblast-Independent Pathway
Authors: Jiang Yanling , Mehta Chetan K. , Hsu Tun-Yi , Alsulaimani Fahad F. H.
Published: July 2002

Bacteria or their products may cause chronic inflammation and subsequent bone loss. This inflammation and bone loss may be associated with significant morbidity in chronic otitis media, periodontitis, endodontic lesions, and loosening of orthopedic implants caused by lipopolysaccharide (LPS)-contaminated implant particles. Currently, it is not clear how bacteria or endotoxin-induced bone resorption occurs and what cell types are involved. Here we report thatPorphyromonas gingivalis, a periodontal pathogen, andEscherichia coliLPS induce osteoclastic cell formation from murine leukocytes in the absence of osteoblasts. In contrast, stimulation with parathyroid hormone had no effect. These multinucleated, tartrate-resistant acid phosphatase-positive cells were positive for receptor activator of NF-κB (RANK), the receptor for osteoprotegerin ligand (OPGL), also known as RANK ligand (RANKL). Blocking antibodies demonstrated that their formation was dependent upon expression of OPGL and, to a lesser extent, on tumor necrosis factor alpha. Mononuclear cells represented a significant source of OPGL production. In vivo,P. gingivalisinjection stimulated OPGL expression in both mononuclear leukocytes and osteoblastic cells. Thus, these findings describe a pathway by which bacteria could enhance osteolysis independently of osteoblasts and suggest that the mix of cells that participate in inflammatory and physiologic bone resorption may be different. This may give insight into new targets of therapeutic intervention.
A Parasitic Phase-Specific Adhesin of
Coccidioides immitis
Contributes to the Virulence of This Respiratory Fungal Pathogen
Authors: Hung Chiung-Yu , Yu Jieh-Juen , Seshan Kalpathi R. , Reichard Utz , Cole Garry T.
Published: July 2002

We report the isolation of a
Coccidioides immitis
gene (
SOWgp
) which encodes an immunodominant, spherule outer wall glycoprotein that is presented as a component of a parasitic phase-specific, membranous layer at the cell surface. The open reading frame of the gene from
C. immitis
isolate C735 translates a 422-amino-acid (aa) polypeptide that contains 6 copies of a 41- to 47-residue tandem repeat enriched in proline (20.4 mol%) and aspartate (19.7%). Two additional isolates of
C. immitis
produce SOWgps of different molecular sizes (328 and 375 aa) and show a corresponding difference in the number of tandem repeats (four and five, respectively). The accurate molecular sizes of these proline-rich antigens, as determined by surface-enhanced laser desorption/ionization mass spectrometry, are comparable to the predicted sizes from the translated protein sequences rather than the estimated sizes based on gel-electrophoretic separation. The results of Northern hybridization confirmed that
SOWgp
expression is parasitic phase specific, and immunoblot studies showed that elevated levels of production of this antigen occurred during early spherule development. The recombinant polypeptide (rSOWp) was shown to bind to mammalian extracellular matrix (ECM) proteins in an in vitro assay (laminin > fibronectin > collagen type IV), suggesting that the parasitic cell surface antigen may function as an adhesin. Deletion of the
SOWgp
gene by using a targeted gene replacement strategy resulted in partial loss of the ability of intact spherules to bind to ECM proteins and a significant reduction in virulence of the mutant strain. The wild-type gene was restored in the mutant by homologous recombination, and the revertant strain was shown to be as virulent as the parental isolate in our murine model of coccidioidomycosis. The parasitic cell surface glycoprotein encoded by the
SOWgp
gene appears to function as an adhesin and contributes to the virulence of
C. immitis.
Streptococcus-Zebrafish Model of Bacterial Pathogenesis
Authors: Neely Melody N. , Pfeifer John D. , Caparon Michael
Published: July 2002

Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish,Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that ofS. iniaein farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogenStreptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.
Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate
Authors: Stafslien D. K. , Cleary P. P.
Published: July 2002

A glutathione-
S
-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca
2+
, Mg
2+
, and Mn
2+
but was inhibited by the same concentrations of Zn
2+
. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp
130
-Ala, His
193
-Ala, Asn
295
-Ala, and Ser
512
-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of
Streptococcus pyogenes
(group A streptococci), and recombinant SCPB, from
Streptococcus agalactiae
(group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP
−1
liter
−1
min
−1
.
Dual Control of
sbo-alb
Operon Expression by the Spo0 and ResDE Systems of Signal Transduction under Anaerobic Conditions in
Bacillus subtilis
Authors: Nakano Michiko M. , Zheng Guolu , Zuber Peter
Published: July 2002

The
Bacillus subtilis sbo-alb
operon contains
sboA
, the structural gene for the bacteriocin subtilosin, and the
alb
genes required for subtilosin production. Transcription from the
sbo-alb
promoter is highly induced by oxygen limitation. The transcriptional regulation of the
sbo-alb
operon is under dual control involving the transition state regulator AbrB and the two-component regulatory proteins ResD and ResE.
The N- and C-Terminal Portions of the
Agrobacterium
VirB1 Protein Independently Enhance Tumorigenesis
Authors: Llosa Matxalen , Zupan John , Baron Christian , Zambryski Patricia
Published: July 2002

Genetic transformation of plants by
Agrobacterium tumefaciens
is mediated by a virulence (
vir
)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a
virB1
deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a
virB1
deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other
vir
genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the
virB1
deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to
vir
regulation.
Autodisplay: Functional Display of Active β-Lactamase on the Surface of
Escherichia coli
by the AIDA-I Autotransporter
Authors: Lattemann Claus T. , Maurer Jochen , Gerland Elke , Meyer Thomas F.
Published: July 2002

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the
Escherichia coli
adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme β-lactamase, leading to efficient expression of the fusion protein in
E. coli
. The β-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located β-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of β-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of
E. coli
cells expressing the β-lactamase AIDA-I fusion protein remained physiologically intact.
Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile
Authors: Bera Aloke Kumar , Chen Sihong , Smith Janet L. , Zalkin Howard
Published: July 2002

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from
Aquifex aeolicus
were expressed in
Escherichia coli
, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH
3
from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH
3
for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.
Role of the
Pseudomonas aeruginosa oxyR-recG
Operon in Oxidative Stress Defense and DNA Repair: OxyR-Dependent Regulation of
katB-ankB
,
ahpB
, and
ahpC-ahpF
Authors: Ochsner Urs A. , Vasil Michael L. , Alsabbagh Eyad , Parvatiyar Kislay , Hassett Daniel J.
Published: July 2002

Pseudomonas aeruginosa
possesses an extensive armament of genes involved in oxidative stress defense, including
katB-ankB
,
ahpB
, and
ahpC-ahpF
. Transcription of these genes was regulated in response to H
2
O
2
, paraquat, or organic peroxides. Expression of
katB-lacZ
and the observed KatB catalase levels in
P. aeruginosa
PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also,
ahpB-lacZ
and
ahpC-lacZ
expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 μM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a Δ
oxyR
mutant, indicating that OxyR was essential for this response. The transcriptional start sites of
katB-ankB
,
ahpB
, and
ahpC-ahpF
were mapped, putative OxyR-binding sites were identified upstream of the −35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The
oxyR
mutant was hypersusceptible to oxidative stress-generating agents, including H
2
O
2
and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The
oxyR
phenotype was fully complemented by a plasmid containing the
oxyR
gene, while any of the
katB
,
ahpB
, or
ahpCF
genes alone resulted in only marginal complementation. Increased
katB-lacZ
expression and higher KatB catalase levels were detected in a Δ
ahpCF
background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In
P. aeruginosa
,
oxyR
is located upstream of
recG
, encoding a putative DNA repair enzyme.
oxyR-lacZ
and
recG-lacZ
reporter activities and
oxyR-recG
mRNA analysis showed that
oxyR
and
recG
are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of
oxyR
. Mutants affected in
recG
but not
oxyR
were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the
oxyR-recG
locus is essential for oxidative stress defense and for DNA repair.
Antigen 43 from
Escherichia coli
Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of
Pseudomonas fluorescens
Authors: Kjærgaard Kristian , Schembri Mark A. , Hasman Henrik , Klemm Per
Published: July 2002

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of
Escherichia coli
. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of
E. coli
cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in
E. coli
. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important
Pseudomonas fluorescens
strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between
E. coli
and
P. fluorescens
. Furthermore, Ag43 expression enhances biofilm formation in
P. fluorescens
to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.
The Stringent Response of
Mycobacterium tuberculosis
Is Required for Long-Term Survival
Authors: Primm Todd P. , Andersen Susan J. , Mizrahi Valerie , Avarbock David , Rubin Harvey , Barry Clifton E.
Published: July 2002

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins.
Mycobacterium tuberculosis
contains a single homolog of these proteins (Rel
Mtb
) and responds to nutrient starvation by producing (p)ppGpp. A

rel
Mtb

knockout strain was constructed in a virulent strain of
M. tuberculosis
, H37Rv, by allelic replacement. The

rel
Mtb

mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel
Mtb
-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel
Mtb
-mediated adaptation. Long-term survival of the

rel
Mtb

mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anerobic incubation than the wild-type virulent organism. Thus, the Rel
Mtb
protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.
Lesions in
gshA
(Encoding γ-
l
-Glutamyl-
l
-Cysteine Synthetase) Prevent Aerobic Synthesis of Thiamine in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Gralnick Jeffrey , Webb Eric , Beck Brian , Downs Diana
Published: July 2002

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in
Salmonella enterica
serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in
gshA
, encoding γ-
l
-glutamyl-
l
-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including
S. enterica
LT2. Phenotypic characterization of the
gshA
mutants indicated they were similar enough to
apbC
and
apbE
mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of
l
-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.
Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of
Corynebacterium glutamicum
Authors: Molenaar Douwe , van der Rest Michel E. , Drysch André , Yücel Raif
Published: July 2002

Like many other bacteria,
Corynebacterium glutamicum
possesses two types of
l
-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC
1.1.99.16
) and a cytoplasmic malate dehydrogenase (MDH; EC
1.1.1.37
) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of
C. glutamicum
. A mutant with a site-directed deletion in the
mqo
gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an
mqo
mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from
C. glutamicum
catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an
mdh
deletion alone was observed, which leaves a physiological function for MDH in
C. glutamicum
obscure.
Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of
Escherichia coli
Authors: van der Rest Michel E. , Frank Christian , Molenaar Douwe
Published: July 2002

Oxidation of malate to oxaloacetate in
Escherichia coli
can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC
1.1.1.37
) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC
1.1.99.16
), encoded by the gene
mqo
(previously called
yojH
). Expression of the
mqo
gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the
mqo
gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress
mqo
expression. On the contrary, MQO and MDH are active at the same time in
E. coli
. For
Corynebacterium glutamicum
, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of
E. coli
. In this organism, a defined deletion of the
mdh
gene led to severely decreased rates of growth on several substrates. Deletion of the
mqo
gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an
mqo
mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes
sfcA
and
b2463
(coding for EC
1.1.1.38
and EC
1.1.1.40
, respectively) and of the phosphoenolpyruvate synthase (EC
2.7.9.2
) gene
pps
were created. They were introduced separately or together with the deletion of
mqo
. These studies did not reveal a significant role for MQO in malate oxidation in wild-type
E. coli
. However, comparing growth of the
mdh
single mutant to that of the double mutant containing
mdh
and
mqo
deletions did indicate that MQO partly takes over the function of MDH in an
mdh
mutant.
Subunit Interactions and Glutamine Utilization by
Escherichia coli
Imidazole Glycerol Phosphate Synthase
Authors: Klem Thomas J. , Chen Yuan , Davisson V. Jo
Published: July 2002

A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric
Escherichia coli
imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however,
k
cat
/

K
m

for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.
Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of an
l
-Arginine/γ-Aminobutyric Acid Permease in
Mycobacterium bovis
BCG
Authors: Seth Anjali , Connell Nancy D.
Published: July 2002

Genes encoding
l
-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from
Mycobacterium bovis
BCG and the effects of its deletion on
l
-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the
rocE
gene of
Bacillus subtilis
, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using
14
C-labeled substrates. Greatly reduced uptake of
l
-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),
l
-[
14
C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous
l
-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium.
l
-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.
Molecular Basis for the Temperature Sensitivity of
Escherichia coli pth
(Ts)
Authors: Cruz-Vera L. Rogelio , Toledo Ivonne , Hernández-Sánchez Javier , Guarneros Gabriel
Published: July 2002

The gene
pth
, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of
Escherichia coli
. Two
pth
mutants have been studied in depth: a
pth
(Ts) mutant isolated as temperature sensitive and a
pth
(rap) mutant selected as nonpermissive for bacteriophage λ vegetative growth. Here we show that each mutant protein is defective in a different way. The Pth(Ts) protein was very unstable in vivo, both at 43°C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the
pth
(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43°C. Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced
pth
(Ts) cell viability even at a permissive temperature. Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis.
Multiple Interactions between Pullulanase Secreton Components Involved in Stabilization and Cytoplasmic Membrane Association of PulE
Authors: Possot Odile M. , Vignon Guillaume , Bomchil Natalia , Ebel Frank , Pugsley Anthony P.
Published: July 2002

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from
Klebsiella oxytoca
encoded by a multiple-copy-number plasmid in
Escherichia coli
. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of
Erwinia chrysanthemi
and
Erwinia carotovora
, showing that they do not play a role in secretory protein recognition and secretion specificity.
Intrinsic Polymerase Activities of UmuD′
2
C and MucA′
2
B Are Responsible for Their Different Mutagenic Properties during Bypass of a T-T
cis-syn
Cyclobutane Dimer
Authors: O'Grady Paul I. , Borden Angela , Vandewiele Dominique , Ozgenc Ali , Woodgate Roger , Lawrence Christopher W.
Published: July 2002

In wild-type
Escherichia coli
, translesion replication is largely dependent upon the UmuD′
2
C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA′
2
B. Interestingly, both the efficiency of translesion replication of a T-T
cis-syn
dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of
dnaQ
(ɛ subunit) or
holE
(θ subunit) or carrying the
dnaQ
allele
mutD5
, which is deficient in proofreading but is competent in the structural function of ɛ, or the
dnaE
antimutator allele
spq-2
. The chromosomal copy of the
umuDC
operon was deleted in each strain, and the UmuDC, UmuD′C, MucAB, or MucA′B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA′
2
B complex is more efficient in promoting translesion replication than the UmuD′
2
C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA′
2
B than by UmuD′
2
C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.
Action of RNase II and Polynucleotide Phosphorylase against RNAs Containing Stem-Loops of Defined Structure
Authors: Spickler Catherine , Mackie George A.
Published: July 2002

The 3′→5′ exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3′ extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.