bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Lymphocyte Proliferation Responses following Primary Immunization with Rabies Vaccine as Neoantigen
Authors: Ghaffari Guity , Passalacqua Dominick J. , Bender Bradley S. , Briggs Debora J. , Goodenow Maureen M. , Sleasman John W.
Published: July 2002

Evaluation of the T-cell immune response following primary antigenic challenge with a neoantigen is a critical aspect of assessment of the cellular immune response. While many antigens can be used to accurately assess in vitro T-cell proliferation to a recall antigen, only a few neoantigens have been tested for their capacities to measure T-cell responses in vitro to a primary immunization. Rabies vaccination is an excellent candidate for the testing of T-cell proliferation responses to a primary immunization because few individuals have been exposed to rabies virus antigens. In the present study 14 rabies vaccine-naı̈ve, healthy adult volunteers were immunized against rabies virus, and T-cell proliferation and antibody responses were measured before and after vaccination. Optimal lymphocyte proliferation to soluble rabies virus antigen occurred after 8 days in culture. The average level of uptake of tritiated thymidine postimmunization was 29,620 ± 4,448 cpm, whereas preimmunization levels were 12,660 ± 3,448 cpm (
P
= 0.002). All individuals showed increases in rabies virus antibody titers from <0.05 to 5.59 ± 1.64 IU/ml. The degree of proliferation to tetanus toxoid as a recall antigen was similar to the response to rabies virus antigen among the cohort. Due to high levels of preimmunization proliferation, four subjects failed to demonstrate a twofold increase in response to rabies virus antigen. The high levels of T-cell responses may be due to a viral superantigen effect in some individuals. Rabies vaccination offers a safe and effective means for measurement of both T- and B-cell immune responses to a neoantigen in healthy and immune suppressed individuals.
Biology ofGiardia lamblia
Authors: Adam Rodney D.
Published: July 2002

Giardia lambliais a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from otherGiardiaspecies by light or electron microscopy. The two major genotypes ofG. lambliathat infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal thatGiardiaspp. are among the most primitive eukaryotes.G. lambliaprobably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5′ and 3′ untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from onevspgene to another have not been associated with sequence alterations or gene rearrangements. TheGiardiagenome project promises to greatly increase our understanding of this interesting and enigmatic organism.
Interleukin-1 Receptor Type I Gene-Deficient Mice Are Less Susceptible toStaphylococcus epidermidisBiomaterial-Associated Infection than Are Wild-Type Mice
Authors: Boelens Jaap Jan , van der Poll Tom , Zaat Sebastian A. J. , Murk Jean L. A. N. , Weening Jan J. , Dankert Jacob
Published: July 2002

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected withStaphylococcus epidermidis.To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R−/−) and wild-type mice received subcutaneous implants of silicon elastomer (SE) or polyvinylpyrrolidone-grafted SE (SEpvp), combined with an injection of 106CFU ofS. epidermidisor sterile saline. Neither mouse strain was susceptible to BAI around SE. IL-1R−/−mice with SEpvp implants had a no abscess formation and a reduced susceptibility to persistentS. epidermidisinfection. The normal foreign body response, characterized by giant-cell formation and encapsulation, was delayed around SEpvp in wild-type mice but not in IL-1R−/−mice. This coincided with enhanced local IL-4 production in IL-1R−/−mice. These data suggest that inhibition of local IL-1 activity may be beneficial for the outcome of BAI.
Identification of the Operon for the Sorbitol (Glucitol) Phosphoenolpyruvate:Sugar Phosphotransferase System in
Streptococcus mutans
Authors: Boyd David A. , Thevenot Tracy , Gumbmann Markus , Honeyman Allen L. , Hamilton Ian R.
Published: July 2002

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol
P
-enolpyruvate phosphotransferase transport system (PTS) of
Streptococcus mutans
LT11. The first gene,
srlD
, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by
srlR
, coding for a transcriptional regulator;
srlM
, coding for a putative activator; and the
srlA
,
srlE
, and
srlB
genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the
srlD
gene is found after the genes coding for the EII components; thus, the location of the gene in
S. mutans
is unique. The SrlR protein is similar to several transcriptional regulators found in
Bacillus
spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865–874, 1998), and its gene overlaps the
srlM
gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria.
Enhanced Susceptibility to Subcutaneous Abscess Formation and Persistent Infection around Catheters Is Associated with Sustained Interleukin-1β Levels
Authors: Boelens J. J. , Zaat S. A. J. , Murk J. L. , Weening J. J. , van der Poll T. , Dankert J.
Published: July 2002

A persistentStaphylococcus epidermidisinfection in mice around a subcutaneous polyvinylpyrrolidone-grafted silicon elastomer catheter (SEpvp) but not around a conventional silicon elastomer catheter was observed. With SEpvp pericatheter tissue, protracted and exaggerated interleukin-1β (IL-1β) production was found. Apparently, sustained levels of IL-1β are associated with enhanced susceptibility to biomaterial-associatedS. epidermidisinfection.
Human Dendritic Cells Are Superior to B Cells at Presenting a Major Histocompatibility Complex Class II-Restricted Heterologous Antigen Expressed on RecombinantStreptococcus gordonii
Authors: Corinti Silvia , Medaglini Donata , Prezzi Caterina , Cavani Andrea , Pozzi Gianni , Girolomoni Giampiero
Published: July 2002

Bacteria are being actively investigated as vaccine carriers for inducing or boosting protective immune responses. In this study, human monocyte-derived dendritic cells (DCs) and normal B cells were compared for their capacity to present the C fragment of tetanus toxin (TTFC), expressed on the surface of recombinantStreptococcus gordonii, to specific CD4+T lymphocytes. DCs were more efficient than B cells at presenting soluble TTFC and remarkably more capable of presenting bacterium-associated TTFC both in terms of the amount of antigen required to obtain a given T-cell response and on a per-cell basis. This difference was associated with a much lower capacity of B cells to endocytose soluble TTFC and phagocytose recombinantS. gordonii. In addition,S. gordoniiinduced the phenotypic maturation of DCs but not of B cells. The results thus indicate that DCs but not B cells play a crucial role in the amplification of class II-restricted immune responses induced by immunization with recombinant gram-positive bacteria.
Induction of Apoptotic Cell Death in Peripheral Blood Mononuclear and Polymorphonuclear Cells by an Oral Bacterium,Fusobacterium nucleatum
Authors: Jewett Anahid , Hume Wyatt R. , Le Ho , Huynh Tri N. , Han Yiping W. , Cheng Genhong , Shi Wenyuan
Published: July 2002

It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells. In this paper we present evidence for the immunosuppressive role ofFusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease. Our studies indicate that the immunosuppressive role ofF. nucleatumis largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs).F. nucleatumtreatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays. The ability ofF. nucleatumto induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis. The data also indicated thatF. nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-κB. Possible mechanisms ofF. nucleatum's role in the pathogenesis of periodontal disease are discussed.
Acquisition of Expression of the
Pseudomonas aeruginosa
ExoU Cytotoxin Leads to Increased Bacterial Virulence in a Murine Model of Acute Pneumonia and Systemic Spread
Authors: Allewelt Markus , Coleman Fadie T. , Grout Martha , Priebe Gregory P. , Pier Gerald B.
Published: July 2002

Pseudomonas aeruginosa
is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing
P. aeruginosa
virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by
P. aeruginosa
is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the
exoU
gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the
exoU
gene into noncytotoxic strains of
P. aeruginosa
lacking this gene affected virulence. Seven phenotypically noncytotoxic
P. aeruginosa
strains were transformed with pUCP19
exoUspcU
which carries the
exoU
gene and its associated chaperone. Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera. The 50% lethal dose of
exoU
-expressing strains was significantly lower for all three
P. aeruginosa
isolates carrying plasmid pUCP19
exoUspcU
than for the isogenic
exoU
-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed
P. aeruginosa
strains. Introduction of the
exoU
gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic
P. aeruginosa
strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.
Surfactant Protein D Enhances Phagocytosis and Killing of Unencapsulated Phase Variants ofKlebsiella pneumoniae
Authors: Ofek Itzhak , Mesika Adi , Kalina Moshe , Keisari Yona , Podschun Ranier , Sahly Hany , Chang Donald , McGregor David , Crouch Erika
Published: July 2002

Pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin (collectin) that is secreted into the alveoli and distal airways of the lung. We have studied the interactions of SP-D and alveolar macrophages withKlebsiella pneumoniae, a common cause of nosocomial pneumonia. SP-D does not agglutinate encapsulatedK. pneumoniaebut selectively agglutinates spontaneous, unencapsulated phase variants, such asKlebsiellastrain K50-3OF, through interactions with their lipopolysaccharides (LPS). These effects are calcium dependent and inhibited with maltose but not lactose, consistent with involvement of the SP-D carbohydrate recognition domain. Precoating of K50-3OF with SP-D enhances the phagocytosis and killing of these organisms by rat alveolar macrophages in cell culture and stimulates the production of nitric oxide by the NR-8383 rat alveolar macrophage cell line. SP-D similarly enhances the NO response to K50-3OF LPS adsorbed to Latex beads under conditions where soluble LPS or SP-D, or soluble complexes of SP-D and LPS, do not stimulate NO production. Our studies demonstrate that interactions of SP-D with exposed arrays ofKlebsiellaLPS on a particulate surface can enhance the host defense activities of alveolar macrophages and suggest that activation of macrophages by SP-D requires binding to microorganisms or other particulate ligands. Because unencapsulated phase variants are likely to be responsible for the initial stages of tissue invasion and infection, we speculate that SP-D-mediated agglutination and/or opsonization ofK. pneumoniaeis an important defense mechanism for this respiratory pathogen in otherwise healthy individuals.
TonB-Dependent Systems of UropathogenicEscherichia coli: Aerobactin and Heme Transport and TonB Are Required for Virulence in the Mouse
Authors: Torres Alfredo G. , Redford Peter , Welch Rodney A. , Payne Shelley M.
Published: July 2002

The uropathogenicEscherichia colistrain CFT073 has multiple iron acquisition systems, including heme and siderophore transporters. AtonBmutant derivative of CFT073 failed to use heme as an iron source or to utilize the siderophores enterobactin and aerobactin, indicating that transport of these compounds in CFT073 is TonB dependent. The TonB−derivative showed reduced virulence in a mouse model of urinary tract infection. Virulence was restored when thetonBgene was introduced on a plasmid. To determine the importance of the individual TonB-dependent iron transport systems during urinary tract infections, mutants defective in each of the CFT073 high-affinity iron transport systems were constructed and tested in the mouse model. Mouse virulence assays indicated that mutants defective in a single iron transport system were able to infect the kidney when inoculated as a pure culture but were unable to efficiently compete with the wild-type strain in mixed infections. These results indicate a role for TonB-dependent systems in the virulence of uropathogenicE. colistrains.
Regulatory Effects of Macrophage Inflammatory Protein 1α/CCL3 on the Development of Immunity toCryptococcus neoformansDepend on Expression of Early Inflammatory Cytokines
Authors: Olszewski Michal A. , Huffnagle Gary B. , Traynor Timothy R. , McDonald Roderick A. , Cook Donald N. , Toews Galen B.
Published: July 2002

Macrophage inflammatory protein 1α (MIP-1α)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain ofCryptococcus neoformans. The present study evaluated the interaction of MIP-1α with other innate immune system cytokines by comparing the immune responses that followed pulmonary infections with high- (C. neoformans145A) and low (C. neoformans52D)-virulence strains. In contrast to what was found forC. neoformans145A infection, lack of MIP-1α inC. neoformans52D infection did not cause the development of EP.C. neoformans52D induced tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1α knockout (KO) mice by day 7 postinfection. Both WT and MIP-1α KO mice subsequently cleared this infection. Thus, the robust expression of early inflammatory cytokines inC. neoformans52D-infected mice promoted the development of protective immunity even in the absence of MIP-1α. Alternatively,C. neoformans145A-infected WT and MIP-1α KO mice had diminished TNF-α, IFN-γ, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulentC. neoformans145A evaded early innate host defenses. HoweverC. neoformans145A-infected WT mice had an early induction of MIP-1α and subsequently did not develop EP. In contrast,C. neoformans145A-infected MIP-1α KO mice developed EP and had increasedC. neoformansdissemination into the brain by day 35. We conclude that, in the absence of other innate immune response effector molecules, MIP-1α is crucial to prevent the development of EP and to controlC. neoformansdissemination to the brain.
Molecular Evolution of Large Virulence Plasmid in
Shigella
Clones and Enteroinvasive
Escherichia coli
Authors: Lan Ruiting , Lumb Brad , Ryan David , Reeves Peter R.
Published: July 2002

Three genes,
ipgD, mxiC
, and
mxiA,
all in the invasion region of the
Shigella
virulence plasmid, were sequenced from strains representing a range of
Shigella
serotypes and from two enteroinvasive
Escherichia coli
(EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in
Shigella flexneri
strains F6 and F6A,
S. boydii
strains B1, B4, B9, B10, B14, and B15,
S. dysenteriae
strains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in
S. flexneri
strains F1A, F2A, F3A, F3C, F4A, and FY, two
S. boydii
strains (B11 and B12), and
S. sonnei
. The D1 pINV plasmid is a recombinant with
ipgD
gene more closely related to those of pINV A but with
mxiA
and
mxiC
genes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of
Shigella
strains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567–10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those
Shigella
(D8 and D10 and
S. sonnei
) and EIEC strains which do not group with the main body of
Shigella
strains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.
Endogenous Interleukin-12 Is Not Required for Resolution ofChlamydophila abortus(Chlamydia psittaciSerotype 1) Infection in Mice
Authors: Del Rı́o Laura , Buendı́a Antonio J. , Sánchez Joaquı́n , Gallego Marı́a C. , Caro Marı́a R. , Ortega Nieves , Seva Juan , Pallarés Francisco J. , Cuello Francisco , Salinas Jesús
Published: July 2002

A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminateChlamydophila abortusinfections. In this study, the role of interleukin-12 (IL-12) in protecting againstC. abortusinfection was investigated using IL-12−/−and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/−mice were able to eliminate theC. abortusinfection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/−mice were infected with a sublethal dose ofC. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/−infected mice. Both WT and IL-12−/−mice developed a Th1 immune response againstC. abortusinfection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge withC. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/−mice. The lack of IL-12 resulted in few infiltrating CD4+T cells in the liver relative to the number in WT mice, although the number of CD8+T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance ofC. abortusinfection, this cytokine is not essential for the establishment of a protective host response against the infection.
Detection of Melanin-Like Pigments in the Dimorphic Fungal PathogenParacoccidioides brasiliensisIn Vitro and during Infection
Authors: Gómez Beatriz L. , Nosanchuk Joshua D. , Dı́ez Soraya , Youngchim Sirida , Aisen Philip , Cano Luz E. , Restrepo Angela , Casadevall Arturo , Hamilton Andrew J.
Published: July 2002

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogenParacoccidioides brasiliensisproduce melanin or melanin-like compounds in vitro and during infection. Growth ofP. brasiliensismycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium withl-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro whenP. brasiliensiswas grown inl-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis froml-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected withP. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology ofP. brasiliensisyeasts. These data strongly suggest thatP. brasiliensispropagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.
Isolation and Properties of Stachyrase A, a Chymotrypsin-Like Serine Proteinase from
Stachybotrys chartarum
Authors: Kordula Tomasz , Banbula Agnieszka , Macomson Jeremy , Travis James
Published: July 2002

A strain of the common mold
Stachybotrys chartarum
has been isolated from the lung of a child with pulmonary hemorrhage. We report the purification of stachyrase A, a new serine chymotrypsin-like proteinase from
S. chartarum
. This enzyme cleaves major protease inhibitors, several biologically active peptides, and collagen, all of which are found in the lung.
Gastritis and Hypergastrinemia Due toAcinetobacter lwoffiiin Mice
Authors: Zavros Y. , Rieder G. , Ferguson Amy , Merchant J. L.
Published: July 2002

In mouse models and humans,Helicobacter pyloriis associated with an increase in serum gastrin and gastrin-expressing (G) cells with a concomitant decrease in somatostatin-expressing D cells. Inflammation of the gastric mucosa can progress to metaplastic changes in the stomach and to decreased colonization byH. pyloriand increased colonization by non-H. pyloriorganisms. In addition, about 20% of individuals with chronic gastritis areH. pylorinegative, suggesting that other organisms may induce gastritis. Consistent with this hypothesis, we report here thatAcinetobacter lwoffiicauses the same histologic changes as doesH. pylori. Gastric epithelial cells were isolated from the entire stomach by an enzymatic method for quantitation by both flow cytometry and morphometric analysis. Two months after mice were inoculated withH. pyloriorA. lwoffii, the mucosal T- and B-cell numbers significantly increased. After 4 months of infection, there was a threefold increase in the number of G cells and a doubling in the number of parietal cells. A threefold decrease in the number of D cells occurred inH. pylori-andA. lwoffii-infected mice. Plasma gastrin levels increased after bothH. pyloriandA. lwoffiiinfection. Histology revealed the presence of inflammation in the gastric mucosa with bothA. lwoffiiandH. pyloriinfection. A periodic acid-Schiff stain-alcian blue stain revealed mucous gland metaplasia of the corpus. Collectively, the results demonstrate that gastritis and hypergastrinemia are not specific forH. pyloribut can be induced by other gram-negative bacteria capable of infecting the mouse stomach.
Initiation of Protein Synthesis in
Saccharomyces cerevisiae
Mitochondria without Formylation of the Initiator tRNA
Authors: Li Yan , Holmes William B. , Appling Dean R. , RajBhandary Uttam L.
Published: July 2002

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA
fMet
) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA
fMet
). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses
N
10
-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C
1
-tetrahydrofolate (C
1
-THF) synthase (
MIS1
), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated
FMT1
). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA
fMet
, Met-tRNA
fMet
, and tRNA
fMet
shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective “wild-type” parental strains. In spite of the absence of fMet-tRNA
fMet
, the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of
petite
colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the
mis1
deletion strain but not for the
fmt1
deletion strain.
Entry into and Release of Solvents by
Escherichia coli
in an Organic-Aqueous Two-Liquid-Phase System and Substrate Specificity of the AcrAB-TolC Solvent-Extruding Pump
Authors: Tsukagoshi Norihiko , Aono Rikizo
Published: July 2002

Growth of
Escherichia coli
is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log
P
OW
of the solvent, where
P
OW
is the partition coefficient measured for the partition equilibrium established between the
n
-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of Δ
acrAB
and/or Δ
tolC
mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log
P
OW
= 5.5). The Δ
tolC
mutant was more sensitive to nonane than the Δ
acrAB
mutant. The solvent entered the
E. coli
cells rapidly. Entry of solvents with a log
P
OW
higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The Δ
tolC
mutant accumulated
n
-nonane or decane (log
P
OW
= 6.0) more abundantly than the parent or the Δ
acrAB
mutant. The AcrAB-TolC complex likely extrudes solvents with a log
P
OW
in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and
n
-hexane.
Gene Conservation and Loss in the
mutS-rpoS
Genomic Region of Pathogenic
Escherichia coli
Authors: Herbelin Corinne J. , Chirillo Samantha C. , Melnick Kristen A. , Whittam Thomas S.
Published: July 2002

The extent and nature of DNA polymorphism in the
mutS-rpoS
region of the
Escherichia coli
genome were assessed in 21 strains of enteropathogenic
E. coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) and in 6 strains originally isolated from natural populations. The intervening region between
mutS
and
rpoS
was amplified by long-range PCR, and the resulting amplicons varied substantially in length (7.8 to 14.2 kb) among pathogenic groups. Restriction maps based on five enzymes and sequence analysis showed that strains of the EPEC 1, EPEC 2, and EHEC 2 groups have a long
mutS-rpoS
region composed of a ∼6.0-kb DNA segment found in strain K-12 and a novel DNA segment (∼2.9 kb) located at the 3′ end of
rpoS
. The novel segment contains three genes (
yclC
,
pad1
, and
slyA
) that occur in
E. coli
O157:H7 and related strains but are not found in K-12 or members of the ECOR group A. Phylogenetic analysis of the common sequences indicates that the long intergenic region is ancestral and at least two separate deletion events gave rise to the shorter regions characteristic of the
E. coli
O157:H7 and K-12 lineages.
Cloning, Characterization, and Inactivation of the Gene
pbpC
, Encoding Penicillin-Binding Protein 3 of
Staphylococcus aureus
Authors: Pinho Mariana G. , de Lencastre Hermínia , Tomasz Alexander
Published: July 2002

The gene
pbpC
from
Staphylococcus aureus
was sequenced: it encodes a 691-amino-acid protein with all of the conserved motifs of a class B high-molecular-weight penicillin-binding protein (PBP), including the transpeptidase conserved motifs SXXK, SXN, and KTG. Insertional inactivation of
pbpC
and introduction of the intact gene in a laboratory mutant missing PBP 3 showed that the
pbpC
gene encodes the staphylococcal PBP 3. Inactivation of
pbpC
caused no detectable change in the muropeptide composition of cell wall peptidoglycan and had only minimum, if any, effect on growth rates, but caused a small but significant decrease in rates of autolysis. Cells of abnormal size and shape and disoriented septa were produced when bacteria with inactivated
pbpC
were grown in the presence of a sub-MIC of methicillin.
Escherichia coli
RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25
Authors: Delgado Mónica A. , Rintoul Marı́a R. , Farı́as Ricardo N. , Salomón Raúl A.
Published: July 2002

Escherichia coli
microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the
rpoC
gene, encoding the β′ subunit of RNA polymerase, at 90 min on the
E. coli
genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type
rpoC
gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant
rpoC
gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.
Hop Resistance in the Beer Spoilage Bacterium
Lactobacillus brevis
Is Mediated by the ATP-Binding Cassette Multidrug Transporter HorA
Authors: Sakamoto Kanta , Margolles Abelardo , van Veen Hendrik W. , Konings Wil N.
Published: July 2002

Lactobacillus brevis
is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in
L. brevis
is, at least in part, dependent on the expression of the
horA
gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in
Lactococcus lactis
. To study the role of HorA in hop resistance, HorA was functionally expressed in
L. lactis
as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of
L. lactis
to hop compounds and cytotoxic drugs. Drug transport studies with
L. lactis
cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.
External-pH-Dependent Expression of the Maltose Regulon and
ompF
Gene in
Escherichia coli
Is Affected by the Level of Glycerol Kinase, Encoded by
glpK
Authors: Chagneau Claudia , Heyde Martine , Alonso Sylvie , Portalier Raymond , Laloi Patrick
Published: July 2002

The expression of the maltose system in
Escherichia coli
is regulated at both transcriptional and translational levels by the pH of the growth medium (pHo). With glycerol as the carbon source, transcription of
malT
, encoding the transcriptional activator of the maltose regulon, is weaker in acidic medium than in alkaline medium.
malT
transcription became high, regardless of the pHo, when glycerol-3-phosphate or succinate was used as the carbon source. Conversely,
malT
expression was low, regardless of the pHo, when maltose was used as the carbon source. The increase in
malT
transcription, associated with the pHo, requires the presence of glycerol in the growth medium and the expression of the glycerol kinase (GlpK). Changes in the level of
glpK
transcription had a great effect on
malT
transcription. Indeed, a
glpFKX
promoter-down mutation has been isolated, and in the presence of this mutation,
malT
expression was increased. When
glpK
was expressed from a high-copy-number plasmid, the
glpK
-dependent reduced expression of the maltose system became effective regardless of the pHo. Analysis of this repression showed that a
malTp1 malTp10
promoter, which is independent of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex, was no longer repressed by
glpFKX
amplification. Thus, GlpK-dependent repression of the maltose system requires the cAMP-CRP complex. We propose that the pHo may affect a complex interplay between GlpK, the phosphotransferase-mediated uptake of glucose, and the adenylate cyclase.
Transcription Profiling-Based Identification of
Staphylococcus aureus
Genes Regulated by the
agr
and/or
sarA
Loci
Authors: Dunman P. M. , Murphy E. , Haney S. , Palacios D. , Tucker-Kellogg G. , Wu S. , Brown E. L. , Zagursky R. J. , Shlaes D. , Projan S. J.
Published: July 2002

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the
Staphylococcus aureus
genome, was used to identify open reading frames that are regulated by
agr
and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and
agr
,
sarA
, and
agr sarA
mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an
agr
- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including
hla
(alpha-toxin) and
spa
(protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by
agr
and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the
agr
and the
sarA
locus are more-global transcription regulators than previously realized.
Essential PchG-Dependent Reduction in Pyochelin Biosynthesis of
Pseudomonas aeruginosa
Authors: Reimmann Cornelia , Patel Hiten M. , Serino Laura , Barone Mario , Walsh Christopher T. , Haas Dieter
Published: July 2002

The biosynthetic genes
pchDCBA
and
pchEF
, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the
pchR
regulatory gene on the chromosome of
Pseudomonas aeruginosa
. The 4.6-kb region located downstream of the
pchEF
genes was found to contain three additional, contiguous genes,
pchG
,
pchH
, and
pchI
, probably forming a
pchEFGHI
operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the
Yersinia pestis
and
Y. enterocolitica
proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in
pchG
abolished pyochelin formation, whereas mutations in
pchH
and
pchI
did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a
P. aeruginosa
mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed
pchDCBA
and
pchEFG
genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host
Escherichia coli
expressing
pchDCBA
and
pchEFG
together with the
E. coli entD
gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate,
l
-cysteine, ATP, NADPH, and
S
-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in
P. aeruginosa
.
Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors ofStaphylococcus aureus
Authors: Yarwood Jeremy M. , McCormick John K. , Schlievert Patrick M.
Published: July 2002

We have previously demonstrated that the presence of oxygen is necessary for the production of toxic shock syndrome toxin 1 (TSST-1) byStaphylococcus aureusin vitro. To investigate the mechanism by which oxygen might regulate toxin production, we identified homologs inS. aureusof theBacillus subtilis resDEgenes. The two-component regulatory system encoded byresDE, ResD-ResE, has been implicated in the global regulation of aerobic and anaerobic respiratory metabolism inB. subtilis. We have designated theS. aureushomologssrrAB(staphylococcal respiratory response). The effects ofsrrABexpression on expression of RNAIII (the effector molecule of theagrlocus) and on production of TSST-1 (an exotoxin) and protein A (a surface-associated virulence factor) were investigated. Expression of RNAIII was inversely related to expression ofsrrAB. Disruption ofsrrBresulted in increased levels of RNAIII, while expression ofsrrABintranson a multicopy plasmid resulted in repression of RNAIII transcription, particularly in microaerobic conditions. Disruption ofsrrBresulted in decreased production of TSST-1 under microaerobic conditions and, to a lesser extent, under aerobic conditions as well. Overexpression ofsrrABresulted in nearly complete repression of TSST-1 production in both microaerobic and aerobic conditions. Protein A production by thesrrBmutant was upregulated in microaerobic conditions and decreased in aerobic conditions. Protein A production was restored to nearly wild-type levels by complementation ofsrrABinto the null mutant. These results indicate that the putative two-component system encoded bysrrAB, SrrA-SrrB, acts in the global regulation of staphylococcal virulence factors, and may repress virulence factors under low-oxygen conditions. Furthermore,srrABmay provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of virulence factors inS. aureus.
Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745
Authors: Galli Dominique M. , Chen Jinbiao , Novak Karen F. , Leblanc Donald J.
Published: July 2002

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from
Actinobacillus actinomycetemcomitans
, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different
A. actinomycetemcomitans
strains. However, transfer of pVT745 derivatives from
A. actinomycetemcomitans
to
Escherichia coli
JM109 by conjugation was successful only when an
E. coli
origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (
Haemophilus
-
Actinobacillus
-
Pasteurella
) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous
A. actinomycetemcomitans
strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.
N
-Acyl-
l
-Homoserine Lactone-Mediated Regulation of the Lip Secretion System in
Serratia liquefaciens
MG1
Authors: Riedel Kathrin , Ohnesorg Thomas , Krogfelt Karen A. , Hansen Thomas S. , Omori Kenji , Givskov Michael , Eberl Leo
Published: July 2002

The analysis of
Serratia liquefaciens
MG1 ′
luxAB
insertion mutants that are responsive to
N
-butanoyl-
l
-homoserine lactone revealed that expression of
lipB
is controlled by the
swr
quorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.
Dissimilatory Fe(III) and Mn(IV) Reduction by
Shewanella putrefaciens
Requires
ferE
, a Homolog of the
pulE
(
gspE
) Type II Protein Secretion Gene
Authors: DiChristina Thomas J. , Moore Charles M. , Haller Carolyn A.
Published: July 2002

Shewanella putrefaciens
strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb
S. putrefaciens
wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468–1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (
ferE
) whose translated product displayed 87% sequence similarity to
Aeromonas hydrophila
ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type
ferE
with an insertionally inactivated
ferE
construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking
ferE
revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (
pulC
to -
N
homologs) was found in the unannotated genome sequence of
Shewanella oneidensis
(formerly
S. putrefaciens
) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer
+
) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer
−
) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of
S. putrefaciens
proteins involved in anaerobic respiration on Fe(III) and Mn(IV).
Intramolecular Regulation of the Opposing (p)ppGpp Catalytic Activities of Rel

Seq

, the Rel/Spo Enzyme from
Streptococcus equisimilis
Authors: Mechold Undine , Murphy Helen , Brown Larissa , Cashel Michael
Published: July 2002

Catalytic and regulatory domains of the Rel/Spo homolog of
Streptococcus equisimilis
affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel

Seq

protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3′-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn
2+
-dependent 3′-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel

Seq

to mediate (p)ppGpp accumulation during amino acid starvation in
S. equisimilis
is absent when the protein is expressed ectopically in
Escherichia coli.
Fusing the N-terminal half of Rel

Seq

with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in
E. coli
by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.