bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Trait Loci Controlling Vegetative Growth Rate in the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Idareta Eneko , Arana Dani , Ritter Enrique , Pisabarro Antonio G. , Ramírez Lucia
Published: July 2002

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete
Pleurotus ostreatus
growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.
Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus
Authors: Smith Jeffrey G. , Liu Xu , Kaufhold Robin M. , Clair James , Caulfield Michael J.
Published: July 2002

Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4+T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.
Early Events in Macrophage Killing of
Aspergillus fumigatus
Conidia: New Flow Cytometric Viability Assay
Authors: Marr Kieren A. , Koudadoust Michael , Black Michele , Balajee S. Arunmozhi
Published: July 2002

Detailed investigations of macrophage phagocytosis and killing of
Aspergillus fumigatus
conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active
A. fumigatus
conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.
Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci
Authors: Russell Nina D. , Corvalan Jose R. F. , Gallo Michael L. , Davis C. Geoffrey , Pirofski Liise-anne
Published: July 2002

Infections withStreptococcus pneumoniaeremain a significant cause of morbidity and mortality. To gain insight into structure-function relationships for human antibodies to pneumococcal capsular polysaccharide (PPS), we studied the response of transgenic mice reconstituted with human immunoglobulin loci, XenoMouse, to PPS antigens in a pneumococcal vaccine. Enzyme-linked immunosorbent assays of sera from mice vaccinated with a 23-valent pneumococcal vaccine revealed that they produced serotype-specific human antibodies, with the greatest response being to the PPS of serotype 3 (PPS 3). Molecular sequence analysis of three monoclonal antibodies (MAbs) to PPS 3 generated from lymphoid cells from mice vaccinated with a 23-valent pneumococcal vaccine or a PPS 3-bovine serum albumin conjugate revealed that they all used heavy-chain immunoglobulin genes from the VH3 family, two expressed light chain genes from the human Vκ1 family, and one expressed a mouse λ light chain. The protective efficacy of the two MAbs was examined in mice. A 10-μg dose of both, and a 1-μg dose of one, significantly prolonged survival from a lethal serotype 3 infection in CBA/N mice. Our data show that XenoMouse mice produced protective, serotype-specific human antibodies to PPS 3, and they lend support to the proposal that these animals represent a useful model to study the human antibody response to PPS antigens.
Helicobacter pyloriModulates Lymphoepithelial Cell Interactions Leading to Epithelial Cell Damage through Fas/Fas Ligand Interactions
Authors: Wang Jide , Fan Xuejun , Lindholm Catharina , Bennett Michael , O'Connoll Joe , Shanahan Fergus , Brooks Edward G. , Reyes Victor E. , Ernst Peter B.
Published: July 2002

Helicobacter pyloricauses a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apoptosis of the epithelium by a Fas/Fas ligand (FasL) interaction. Fas receptor expression was detected on freshly isolated gastric epithelial cells by flow cytometry and immunohistochemistry, and this level of expression was increased during infection withH. pylori. The expression of Fas receptor on three gastric epithelial cell lines was increased byH. pylori, either alone or in combination with gamma interferon or tumor necrosis factor alpha. The role of Fas in apoptosis of gastric epithelial cell lines was evidenced by DNA fragmentation after cross-linking of Fas with specific antibodies. FasL expression was detected by immunohistochemistry on mononuclear cells in gastric biopsy specimens of infected but not uninfected subjects. Gastric T-cell lines were also shown to express FasL, as evidenced by reverse transcription-PCR and killing of target cells expressing Fas receptor. Moreover, these T-cell lines were capable of killing cultured gastric epithelial target cells and antibodies that block the interaction between Fas receptor and FasL inhibited this cytotoxic activity. These observations demonstrate that local Th1 cells may contribute to the pathogenesis of gastric disease duringH. pyloriinfection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions.
Depressed Interleukin-12 (IL-12), but not IL-18, Production in Response to a 30- or 32-Kilodalton Mycobacterial Antigen in Patients with Active Pulmonary Tuberculosis
Authors: Song Chang-Hwa , Kim Hwa-Jung , Park Jeong-Kyu , Lim Jae-Hyun , Kim Un-Ok , Kim Jun-Sang , Paik Tae-Hyun , Kim Kyung-Jin , Suhr Ji-Won , Jo Eun-Kyeong
Published: July 2002

The secreted 30-kDa antigen (Ag) ofMycobacterium tuberculosisdirectly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-γ), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-γ and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.
Vacuole Acidification Is Not Required for Survival ofSalmonella entericaSerovar Typhimurium within Cultured Macrophages and Epithelial Cells
Authors: Steele-Mortimer Olivia , St-Louis Maryse , Olivier Martin , Finlay B. Brett
Published: July 2002

Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellularSalmonella entericaserovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential forSalmonellaintracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.
Shiga Toxin Translocation across Intestinal Epithelial Cells Is Enhanced by Neutrophil Transmigration
Authors: Hurley Bryan P. , Thorpe Cheleste M. , Acheson David W. K.
Published: July 2002

Shiga toxin-producing
E. coli
(STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lacking
eae
and
espB
(
eae
- and
espB
-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion than
eae
- and
espB
-positive STEC or enteropathogenic
E. coli
. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.
Characterization of
sarR
, a Modulator of
sar
Expression in
Staphylococcus aureus
Authors: Manna Adhar , Cheung Ambrose L.
Published: July 2002

The expression of virulence determinants in
Staphylococcus aureus
is controlled by global regulatory loci (e.g.,
sar
and
agr
). The
sar
locus is composed of three overlapping transcripts (
sar
P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp
sarA
gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of
sar
promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column containing a
sar
P2 promoter fragment. In this study, the putative gene, designated
sarR
, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the
sarR
gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound
sar
P1, P2, and P3 promoter fragments in gel shift and footprinting assays. A
sarR
mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant
sar
transcript, we confirmed that the
sarR
mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the
sar
promoters to down-regulate P1 transcription and the ensuing SarA protein expression.
Human Endothelial Cell Activation and Mediator Release in Response toListeria monocytogenesVirulence Factors
Authors: Rose Frank , Zeller Sven-Arne , Chakraborty Trinad , Domann Eugen , Machleidt Thomas , Kronke Martin , Seeger Werner , Grimminger Friedrich , Sibelius Ulf
Published: July 2002

The interaction ofListeria monocytogeneswith endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-typeL. monocytogenes(EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGDhlymutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca2+suggested LLO-elicited Ca2+flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGDhlymutant), listerial phosphatidylinositol-specific phospholipase C (EGDplcAmutant), broad-spectrum phospholipase C (EGDplcBmutant) and internalin B (EGDinlBmutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-κB to the stimulation with the LLO-producing strains EGD andListeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-κB activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-κB translocation and the upregulation of cytokine synthesis. We conclude thatL. monocytogenesis a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca2+flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-κB are suggested as underlying host signaling events. These endothelial responses toL. monocytogenesmay well contribute to the pathogenic sequelae in severe listerial infection and sepsis.
Long-Range Mapping of the
Streptococcus agalactiae
Phylogenetic Lineage Restriction Digest Pattern Type III-3 Reveals Clustering of Virulence Genes
Authors: Bohnsack John F. , Whiting April A. , Bradford Russell D. , Van Frank Brenna K. , Takahashi Shinji , Adderson Elisabeth E.
Published: July 2002

Human isolates of serotype III
Streptococcus agalactiae
(group B streptococcus [GBS]) can be divided into three separate phylogenetic lineages based on analysis of the restriction digest patterns (RDPs) of chromosomal DNA. Nine DNA sequences that are present in all isolates of the RDP III-3 phylogenetic lineage, but not in the other lineages, were identified by genomic subtractive hybridization. A complete physical map of a III-3 chromosome was constructed. Six of the nine III-3-specific sequences mapped to a 340-kb
Sse
8387I fragment which contains or is located close to known GBS virulence genes. One of the III-3-specific probes, AW-10, encodes part of GBSi1, a group II intron that is inserted at two sites within the GBS genome. The second chromosomal site for GBSi1 was isolated, sequenced, and mapped to a location near the locus responsible for hemolysin production. These findings suggest that the genetic variation that distinguishes the RDP type III-3 strains from other serotype III strains occurs largely within localized areas of the genome containing known or putative virulence genes.
Insertional Inactivation of
eap
in
Staphylococcus aureus
Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts
Authors: Hussain Muzaffar , Haggar Axana , Heilmann Christine , Peters Georg , Flock Jan-Ingmar , Herrmann Mathias
Published: July 2002

To initiate invasive infection,
Staphylococcus aureus
must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of
S. aureus
strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable
eap
::
ermB
deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (
P
< 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (
P
< 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.
Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the
Plasmodium falciparum
Apical Membrane Antigen 1 Expressed in
Escherichia coli
Authors: Dutta Sheetij , Lalitha P. V. , Ware Lisa A. , Barbosa Arnoldo , Moch J. Kathleen , Vassell Meredith A. , Fileta Bader B. , Kitov Svetlana , Kolodny Nelly , Heppner D. Gray , Haynes J. David , Lanar David E.
Published: July 2002

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83
Gly
to 531
Glu
) of
Plasmodium falciparum
(3D7) produced in
Escherichia coli.
A synthetic gene containing an
E. coli
codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified
E. coli
strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the
P. falciparum
homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.
Identification of an Effector Specificity Subregion within the Aromatic-Responsive Regulators DmpR and XylR by DNA Shuffling
Authors: Skärfstad Eleonore , O'Neill Eric , Garmendia Junkal , Shingler Victoria
Published: July 2002

The
Pseudomonas
derived ς
54
-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.
Mre11 and Rad50 from
Pyrococcus furiosus
: Cloning and Biochemical Characterization Reveal an Evolutionarily Conserved Multiprotein Machine
Authors: Hopfner Karl-Peter , Karcher Annette , Shin David , Fairley Cecilia , Tainer John A. , Carney James P.
Published: July 2002

The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon
Pyrococcus furiosus
and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the
P. furiosus
Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.
Covalent Linkage of Polyamines to Peptidoglycan in
Anaerovibrio lipolytica
Authors: Hirao Takashi , Sato Masahide , Shirahata Akira , Kamio Yoshiyuki
Published: July 2002

Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of
Anaerovibrio lipolytica
, a strictly anaerobic bacterium. The peptidoglycan was degraded with the
N
-acetylmuramyl-
l
-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified as
l
-alanine–
d
-glutamic acid(
α
cadaverine)
γ
meso
-diaminopimelic acid (DAP)–
d
-alanine and
l
-alanine–
d
-glutamic acid(
α
spermidine)
γ
meso
-DAP–
d
-alanine, respectively. The N
1
-amino group of spermidine was linked to the α-carboxyl group of the
d
-glutamic acid residue of peptide II.
Genetic Evidence of Distinct Physiological Regulation Mechanisms in the ς
54
Pu
Promoter of
Pseudomonas putida
Authors: Cases Ildefonso , de Lorenzo Víctor
Published: July 2002

The activity of the toluene-responsive ς
54
Pu
promoter of the pWW0 TOL plasmid of
Pseudomonas putida
is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. Although the
Pu
promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of β-galactosidase in a
Pu-lacZ
reporter
P. putida
strain. Since
Pu
was still down-regulated during exponential growth regardless of glucose addition, it appeared that the carbohydrate separately influenced promoter activity. This notion was supported by the growth-dependent induction pattern of
Pu
in a
ptsN
mutant of
P. putida
, the loss of which makes
Pu
no longer responsive to repression by glucose. On the other hand, overexpression of the sigma factor ς
54
, known to partially alleviate the exponential silencing of the promoter, did not affect glucose inhibition of
Pu
. These data indicated that exponential silencing and carbon source-dependent repression are two overlapping but genetically distinguishable mechanisms that adapt
Pu
to the physiological status of the cells and nutrient availability.
Methionine-to-Cysteine Recycling in
Klebsiella aerogenes
Authors: Seiflein Thomas A. , Lawrence Jeffrey G.
Published: July 2002

In the enteric bacteria
Escherichia coli
and
Salmonella enterica
, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium
Klebsiella aerogenes
appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where
cys
mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in
E. coli
and
Salmonella
appear to be more intertwined in
Klebsiella
.
Identification of the Domains of UreR, an AraC-Like Transcriptional Regulator of the Urease Gene Cluster in
Proteus mirabilis
Authors: Poore Carrie A. , Coker Christopher , Dattelbaum Jonathan D. , Mobley Harry L. T.
Published: July 2002

Proteus mirabilis
urease catalyzes the hydrolysis of urea to CO
2
and NH
3
, resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins,
ureDABCEFG
, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR (amino acids 164 to 293) activated transcription from the
ureD
promoter (p

ureD

) and bound to a 60-bp fragment containing p

ureD

, as analyzed by gel shift. These results were consistent with the DNA-binding specificity residing in the C-terminal half of UreR and dimerization being required for activity. To localize the dimerization domain of UreR, a translational fusion of the DNA-binding domain of the LexA repressor (amino acids 1 to 87) and the N-terminal half of UreR (amino acids 1 to 182) was constructed and found to repress transcription from p

sulA

-
lac
Z (
sulA
is repressed by LexA) and bind to the
sulA
operator site, as analyzed by gel shift. Since LexA binds this site only as a dimer, the UreR
1–182
-LexA
1–87
fusion also must dimerize to bind p

sulA

. Indeed, purified UreR-
Myc
-His eluted from a gel filtration column as a dimer. Therefore, we conclude that the dimerization domain of UreR is located within the N-terminal half of UreR. UreR contains three leucines that mimic the leucines that contribute to dimerization of AraC. Mutagenesis of Leu147, Leu148, or L158 alone did not significantly affect UreR function. In contrast, mutagenesis of both Leu147 and Leu148 or all three Leu residues resulted in a 85 or 94% decrease, respectively, in UreR function in the presence of urea (
P
< 0.001). On the contrary, His102 and His175 mutations of UreR resulted in constitutive induction in the absence of urea. We conclude that a dimerization domain resides in the N-terminal half of the polypeptide, that Leu residues may contribute to this function, and that sequences within the C-terminal half of UreR are responsible for DNA binding to the urease promoter regions. Selected His residues also contribute significantly to UreR function.
Gonococcal MinD Affects Cell Division in
Neisseria gonorrhoeae
and
Escherichia coli
and Exhibits a Novel Self-Interaction
Authors: Szeto Jason , Ramirez-Arcos Sandra , Raymond Claude , Hicks Leslie D. , Kay Cyril M. , Dillon Jo-Anne R.
Published: July 2002

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus
Neisseria gonorrhoeae
contains a
min
operon, and the present study investigates the role of
minD
from this operon. A gonococcal
minD
insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from
N. gonorrhoeae
(MinD
Ng
) in this mutant. Hence, MinD
Ng
is required for maintaining proper cell division and growth in
N. gonorrhoeae
. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD
Ng
is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC
Ng
and MinD
Ng
from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC
Ng
or MinD
Ng
alone did not display noticeable morphological changes. These studies suggest that MinD
Ng
is involved in inhibiting gonococcal cell division, likely in conjunction with MinC
Ng
. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD
Ng
in wild-type
Escherichia coli
led to cell filamentation, while overexpression in an
E. coli minD
mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD
Ng
revealed a novel MinD
Ng
self-interaction. We have also shown by yeast two-hybrid analysis that MinD from
E. coli
interacts with itself and with MinD
Ng
. These results indicate that MinD
Ng
is required for maintaining proper cell division and growth in
N. gonorrhoeae
and suggests that the self-interaction of MinD may be important for cell division site selection across species.
Transcriptional Analysis of the
tet
(P) Operon from
Clostridium perfringens
Authors: Johanesen Priscilla A. , Lyras Dena , Bannam Trudi L. , Rood Julian I.
Published: July 2002

The
Clostridium perfringens
tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes,
tetA
(P) and
tetB
(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible
tetA
(P) and
tetB
(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the
tetA
(P) start codon. Deletion of P3 or alteration of the spacing between the −35 and −10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the
tetA
(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the
tet
(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.
Exchange of Xcp (Gsp) Secretion Machineries between
Pseudomonas aeruginosa
and
Pseudomonas alcaligenes
: Species Specificity Unrelated to Substrate Recognition
Authors: de Groot Arjan , Koster Margot , Gérard-Vincent Manon , Gerritse Gijs , Lazdunski Andrée , Tommassen Jan , Filloux Alain
Published: July 2002

Pseudomonas aeruginosa
and
Pseudomonas alcaligenes
are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12
xcp
gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with
Erwinia
, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175–190, 1996). In the present study, we report that XcpP and XcpQ of
P. alcaligenes
could not substitute for their respective
P. aeruginosa
counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when
P. alcaligenes xcpP
and
xcpQ
were expressed simultaneously in a
P. aeruginosa xcpPQ
deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable
P. alcaligenes
XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).
Myxococcus xanthus mokA
Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance
Authors: Kimura Yoshio , Nakano Hiromi , Terasaka Hideaki , Takegawa Kaoru
Published: July 2002

A gene,
mokA
, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a
Myxococcus xanthus
genomic library. The predicted
mokA
gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain.
mokA
mutants placed under starvation conditions exhibited reduced sporulation. Mutation of
mokA
also caused marked growth retardation at high osmolarity. These results indicated that
M. xanthus
MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold
Dictyostelium discoideum.
Sequence Diversity and Molecular Evolution of the Leukotoxin (
lktA
) Gene in Bovine and Ovine Strains of
Mannheimia
(
Pasteurella
)
haemolytica
Authors: Davies Robert L. , Whittam Thomas S. , Selander Robert K.
Published: July 2002

The molecular evolution of the leukotoxin structural gene (
lktA
) of
Mannheimia
(
Pasteurella
)
haemolytica
was investigated by nucleotide sequence comparison of
lktA
in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in
M. haemolytica
. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the
lktA
gene of the two related species
Mannheimia glucosida
and
Pasteurella trehalosi
, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess
lktA
alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of
lktA
in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.
Comparative Sequence Analysis of the Symbiosis Island ofMesorhizobium lotiStrain R7A
Authors: Sullivan John T. , Trzebiatowski Jodi R. , Cruickshank Ruth W. , Gouzy Jerome , Brown Steven D. , Elliot Rachel M. , Fleetwood Damien J. , McCallum Nadine G. , Rossbach Uwe , Stuart Gabriella S. , Weaver Julie E. , Webby Richard J. , de Bruijn Frans J. , Ronson Clive W.
Published: July 2002

TheMesorhizobium lotistrain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them toLotussymbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island ofM. lotistrain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include atrboperon and a cluster of potentialtragenes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to thevirpilus fromAgrobacterium tumefaciensthat is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.
An Adenylyl Cyclase, CyaA, of
Myxococcus xanthus
Functions in Signal Transduction during Osmotic Stress
Authors: Kimura Yoshio , Mishima Yukako , Nakano Hiromi , Takegawa Kaoru
Published: July 2002

An adenylyl cyclase gene (
cyaA
) present upstream of an osmosensor protein gene (
mokA
) was isolated from
Myxococcus xanthus. cyaA
encoded a polypeptide of 843 amino acids with a predicted molecular mass of 91,187 Da. The predicted
cyaA
gene product had structural similarity to the receptor-type adenylyl cyclases that are composed of an amino-terminal sensor domain and a carboxy-terminal catalytic domain of adenylyl cyclase. In reverse transcriptase PCR experiments, the transcript of the
cyaA
gene was detected mainly during development and spore germination. A
cyaA
mutant, generated by gene disruption, showed normal growth, development, and germination. However, a
cyaA
mutant placed under conditions of ionic (NaCl) or nonionic (sucrose) osmostress exhibited a marked reduction in spore formation and spore germination. When wild-type and
cyaA
mutant cells at developmental stages were stimulated with 0.2 M NaCl or sucrose, the mutant cells increased cyclic AMP accumulation at levels similar to those of the wild-type cells. In contrast, the mutant cells during spore germination had mainly lost the ability to respond to high-ionic osmolarity. In vegetative cells, the
cyaA
mutant responded normally to osmotic stress. These results suggested that
M. xanthus
CyaA functions mainly as an ionic osmosensor during spore germination and that CyaA is also required for osmotic tolerance in fruiting formation and sporulation.
Inactivation of the Spirochete
recA
Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology
Authors: Kameni Ange-Patricia Tchamedeu , Couture-Tosi Evelyne , Saint-Girons Isabelle , Picardeau Mathieu
Published: July 2002

Recently, we have shown the first evidence for allelic exchange in
Leptospira
spp. By using the same methodology, the cloned
recA
of
Leptospira biflexa
was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the
L. biflexa
chromosome by homologous recombination. The
recA
double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the
recA
mutant was about 10% of that of the parent strain. In addition, microscopic observation of the
L. biflexa recA
mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the
L. biflexa
wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the
recA
mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that
L. biflexa
RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.
Identification of an Acetoacetyl Coenzyme A Synthetase-Dependent Pathway for Utilization of
l
-(+)-3-Hydroxybutyrate in
Sinorhizobium meliloti
Authors: Aneja Punita , Dziak Renata , Cai Guo-Qin , Charles Trevor C.
Published: July 2002

d
-(−)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of
Sinorhizobium meliloti
. In contrast, wild-type
S. meliloti
is not able to utilize the
l
-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the
S. meliloti acsA2
gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in
S. meliloti
is AcsA2 dependent.
Identification of XcpZ Domains Required for Assembly of the Secreton of
Pseudomonas aeruginosa
Authors: Robert Viviane , Hayes Finbarr , Lazdunski Andrée , Michel Gérard P. F.
Published: July 2002

Most of the exoproteins secreted by
Pseudomonas aeruginosa
are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.