bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cultivation of Pathogenic and Opportunistic Free-Living Amebas
Authors: Schuster Frederick L. 
Published: July 2002

SUMMARYFree-living amebas are widely distributed in soil and water, particularly members of the generaAcanthamoebaandNaegleria. Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts.Naegleriais the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis;Acanthamoebais responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis.Balamuthiasp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples.NaegleriaandAcanthamoebacan be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source.Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba,Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.
Cultivation of Clinically Significant Hemoflagellates
Authors: Schuster Frederick L. , Sullivan James J. 
Published: July 2002

SUMMARY

The hemoflagellates,
Trypanosoma
spp. and
Leishmania
spp., are causal agents of a number of parasitic diseases having a major impact on humans and domestic animals over vast areas of the globe. Among the diseases are some of the most pernicious and deadly of human afflictions: African sleeping sickness, Chagas' disease, kala-azar, and Oriental sore. The organisms have complex, pleomorphic life cycles typically involving a vertebrate and an invertebrate host, the latter serving as a vector. In the vertebrate host, they are primarily blood and tissue parasites. In their transition from one host to another, the hemoflagellates undergo morphological, physiological, and biochemical changes that facilitate their growth and subsequent transmission. A major goal in the study of the hemoflagellates has been the cultivation in vitro of both vertebrate and invertebrate stages of the organisms. The first types of media used in their cultivation, and still useful for establishment of cultures, were undefined and contained a complex of ingredients. These gave way to semidefined formulations which included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth. More recently developed media are completely defined, having replaced the feeder cells with various supplements. Serum, a sometimes-variable component of the media, can be replaced by various serum substitutes. This review focuses on the hemoflagellates that infect humans, describing stages in the development of media leading to the fully defined formulations that are now available for the cultivation of many of these organisms.
Rickettsia rickettsiiInfection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression
Authors: Rydkina Elena , Sahni Abha , Silverman David J. , Sahni Sanjeev K. 
Published: July 2002

ABSTRACTExisting evidence suggests that oxidative insults and antioxidant defense mechanisms play a critical role in the host cell response during infection of endothelial cells byRickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Heme oxygenase (HO), a rate-limiting enzyme in the pathway for heme catabolism, protects against oxidant damage in a variety of stress situations. Here, we report on the expression of the inducible and constitutive HO isozymes, HO-1 and HO-2, duringR. rickettsiiinfection of endothelial cells. Steady-state levels for HO-1 mRNA were increased two- to threefold, as early as 4 h postinfection, whereas HO-2 mRNA was not affected. Induction of HO-1 mRNA was dependent on the dose of infection and occurred in a time-dependent manner, reaching maximal levels at 4 to 7 h. The increase in HO-1 mRNA occurred at the level of trancription as it was blocked by the transcriptional inhibitors, actinomycin D and α-amanitin. The eukaryotic protein synthesis inhibitor, cycloheximide, caused a >50% reduction in the infection-induced increase in HO-1 mRNA level, suggesting its dependence on de novo protein synthesis of host cell. The uptake of viable organisms appeared to be necessary, since inactivation ofR. rickettsiiby heat or formalin fixation, or incubation of cells with cytochalasin B to prevent entry resulted in marked inhibition of HO-1 response.N-Acetyl-l-cysteine, a known oxidant scavenger, inhibited the HO-1 induction byR. rickettsii.Finally, Western analysis with a specific monoclonal antibody revealed higher levels of HO-1 protein (∼32 kDa), confirming that changes in HO-1 mRNA levels were followed by increases in the levels of protein. The findings indicate thatR. rickettsiiinfection induces HO-1 expression in host endothelial cells and suggest an important role for this enzyme in cellular response to infection, possibly by serving a protective function against oxidative injury.
Eighty-Kilodalton N-Terminal Moiety of
Bordetella pertussis
Filamentous Hemagglutinin: Adherence, Immunogenicity, and Protective Role
Authors: Alonso Sylvie , Reveneau Nathalie , Pethe Kévin , Locht Camille 
Published: July 2002

ABSTRACT

Bordetella pertussis
, the etiological agent of whooping cough, produces a number of factors, such as toxins and adhesins, that are required for full expression of virulence. Filamentous hemagglutinin (FHA) is the major adhesin of
B. pertussis
. It is a protein of approximately 220 kDa, found both associated at the bacterial cell surface and secreted into the extracellular milieu. Despite its importance in
B. pertussis
pathogenesis and its inclusion in most acellular pertussis vaccines, little is known about the functional importance of individual domains in infection and in the induction of protective immunity. In this study, we analyzed the role of the approximately 80-kDa N-terminal domain of FHA, designated Fha44, in
B. pertussis
adherence, colonization, and immunogenicity. Although Fha44 contains the complete heparan sulfate-binding domain, it is not sufficient for adherence to epithelial cells or macrophages. It also cannot replace FHA during colonization of the mouse respiratory tract. Infection with a
B. pertussis
strain producing Fha44 instead of FHA does not induce anti-FHA antibodies, whereas such antibodies can readily be induced by intranasal administration of purified Fha44. In addition, mice immunized with purified Fha44 were protected against challenge with wild-type
B. pertussis
, indicating that Fha44 contains protective epitopes. Compared to FHA, Fha44 is much smaller and much more soluble and is therefore easier to purify and to store. These advantages may perhaps warrant considering Fha44 for inclusion in acellular pertussis vaccines.
Yersiniabactin Production Requires the Thioesterase Domain of HMWP2 and YbtD, a Putative Phosphopantetheinylate Transferase
Authors: Bobrov Alexander G. , Geoffroy Valerie A. , Perry Robert D. 
Published: July 2002

ABSTRACT

One requirement for the pathogenesis of
Yersinia pestis
, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the
pgm
locus of the
Y. pestis
chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the
pgm
locus. We have identified a gene,
ybtD
, encoded outside the HPI and
pgm
locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of
Escherichia coli.
A deletion within
ybtD
yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from Ybt-producing strains of
Y. pestis.
The promoter region of
ybtD
was fused to
lacZ
; β-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the
ybt
system. The
ybtD
mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other
ybt
biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any
ybt
regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.
Orientia tsutsugamushiInhibits Apoptosis of Macrophages by Retarding Intracellular Calcium Release
Authors: Kim Mee-Kyung , Seong Seung-Yong , Seoh Ju-Young , Han Tae-Hee , Song Hyeon-Je , Lee Ji-Eun , Shin Jung-Hoon , Lim Byung-Uk , Kang Jae-Seung 
Published: July 2002

ABSTRACTOrientia tsutsugamushishows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited byO. tsutsugamushiinfection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected withO. tsutsugamushi. Antiapoptotic activities ofO. tsutsugamushiin infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.
Vibrio Pathogenicity Island and Cholera Toxin Genetic Element-Associated Virulence Genes and Their Expression in Non-O1 Non-O139 Strains of
Vibrio cholerae
Authors: Sarkar Amit , Nandy Ranjan K. , Nair G. Balakrish , Ghose Asoke C. 
Published: July 2002

ABSTRACT

A non-O1 non-O139
Vibrio cholerae
strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259
tcpA
gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (
aldA
,
tagA
,
tcpP/H
,
toxT
,
acfB/C
, and
int
) and intergenic regions (
tcpF
to
toxT
and
tcpH
to
tcpA
) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of
tcpP/H
,
toxR
,
toxT
, and
tcpA
genes, but not of
ctxA
, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of
ctxAB
. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a
V. cholerae
O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of
toxT
,
ctxA
, and
tcpA
when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (∼1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of
toxT
,
ctxA
, or
tcpA
genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of
V. cholerae
strains belonging to non-O1 serogroups.
Evidence from Terminal Recombination Gradients that FtsK Uses Replichore Polarity To Control Chromosome Terminus Positioning at Division inEscherichia coli
Authors: Corre Jacqueline , Louarn Jean-Michel 
Published: July 2002

ABSTRACTChromosome dimers inEscherichia coliare resolved at thediflocus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein. Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires thedifresolution site to be located at the junction between the two polarized chromosome arms or replichores. Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination). We had proposed thatdifsites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes. To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in anftsKmutant. The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on axerCmutant chromosome with respect to intensity, the region affected, and response to polarity distortion. The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localizedifat the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material.
The Glycolytic Flux in
Escherichia coli
Is Controlled by the Demand for ATP
Authors: Koebmann Brian J. , Westerhoff Hans V. , Snoep Jacky L. , Nilsson Dan , Jensen Peter R. 
Published: July 2002

ABSTRACT

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F
1
part of the membrane-bound (F
1
F
0
) H
+
-ATP synthase were expressed in steadily growing
Escherichia coli
cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of
E. coli
. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly “carbon” limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.
Group I Self-Splicing Intron in the
recA
Gene of
Bacillus anthracis
Authors: Ko Minsu , Choi Hyang , Park Chankyu 
Published: July 2002

ABSTRACT

Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the
recA
gene of
Bacillus anthracis
which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the
recA
precursor mRNA for RecA that was being translated in
E. coli
. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of
B. anthracis
expressed in
E. coli
was functional in its ability to complement a
recA
defect. When
recA
-negative
E. coli
cells were irradiated with UV, the
Bacillus
RecA reduced the UV susceptibility of the
recA
mutant, regardless of the presence of intron.
Export of
l
-Isoleucine from
Corynebacterium glutamicum
: a Two-Gene-Encoded Member of a New Translocator Family
Authors: Kennerknecht Nicole , Sahm Hermann , Yen Ming-Ren , Pátek Miroslav , Saier, Milton H. , Eggeling Lothar 
Published: July 2002

ABSTRACT

Bacteria possess amino acid export systems, and
Corynebacterium glutamicum
excretes
l
-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for
l
-isoleucine export, we have used transposon mutagenesis to isolate mutants of
C. glutamicum
sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated
brnF
encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices.
brnE
is located downstream of
brnF
and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports
l
-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of
l
-leucine and
l
-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of
brnFE
and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in α-proteobacteria but not in other prokaryotes analyzed.
The Gonococcal Fur Regulon: Identification of Additional Genes Involved in Major Catabolic, Recombination, and Secretory Pathways
Authors: Sebastian Shite , Agarwal Sarika , Murphy John R. , Genco Caroline Attardo 
Published: July 2002

ABSTRACT

In this study, we have characterized the in vitro binding of
Neisseria gonorrhoeae
Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in
Escherichia coli
HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including
tonB
,
fur
,
recN
,
secY
,
sodB
,
hemO
,
hmbR
,
fumC
, a hypothetical gene (Fe-S homolog), and the
opa
family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of
tonB
,
fur
,
recN
,
secY
,
sodB
,
hemO
,
hmbR
,
fumC
, the Fe-S homolog gene, and the
opa
gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11
opa
genes (
opaA
through -
K
), we did not detect binding of purified
E. coli
Fur with 8 of the 11
opa
members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the
opa
family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.
Differential Spectrum of Mutations That Activate the
Escherichia coli bgl
Operon in an
rpoS
Genetic Background
Authors: Moorthy Sudha , Mahadevan S. 
Published: July 2002

ABSTRACT

The
bgl
promoter is silent in wild-type
Escherichia coli
under standard laboratory conditions, and as a result, cells exhibit a β-glucoside-negative (Bgl
−
) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl
+
phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus,
bglR
, constitutes the major class of activating mutations identified in laboratory cultures. The
rpoS
-encoded σ
S
, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the
rpoS
status of the cell influences the nature of the mutations that activate the
bgl
promoter, we analyzed spontaneously arising Bgl
+
mutants in

rpoS
+

and
rpoS
genetic backgrounds. We show that the spectrum of activating mutations in
rpoS
cells is different from that in

rpoS
+

cells. Unlike

rpoS
+

cells, where insertions in
bglR
are the predominant activating mutations, mutations in
hns
make up the majority in
rpoS
cells. The physiological significance of these differences is discussed in the context of survival of natural populations of
E. coli
.
Importance of DifferenttfdGenes for Degradation of Chloroaromatics byRalstonia eutrophaJMP134
Authors: Plumeier Iris , Pérez-Pantoja Danilo , Heim Sabina , González Bernardo , Pieper Dietmar H. 
Published: July 2002

ABSTRACTThetfdCIDIEIFI,andtfdDIICIIEIIFIIgene modules of plasmid pJP4 ofRalstonia eutrophaJMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity oftfdI-encoded enzymes was usually higher than that oftfdII-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only onetfdgene module. ThetfdDII-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors ofR. eutrophastrains containing differenttfdgene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to formtrans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.