bluuebunny/crossref_metadata_2025 huggingface dataset:
The Phosphoryl Transfer Domain of UhpB Interacts with the Response Regulator UhpA
Authors: Wright Jesse S. , Kadner Robert J.
Published: July 2002

Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription. UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT. Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione
S
-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of
uhpT
expression in a
uhp
+
strain. This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation. The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation. Point mutations which allow GST-Bc to activate
uhpT
expression or which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain. The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR. The GST-Bc protein accelerated dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313. Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred. Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA.
Reconstitution of Acetosyringone-Mediated
Agrobacterium tumefaciens
Virulence Gene Expression in the Heterologous Host
Escherichia coli
Authors: Lohrke Scott M. , Yang Hongjiang , Jin Shouguang
Published: July 2002

The ability to utilize
Escherichia coli
as a heterologous system in which to study the regulation of
Agrobacterium tumefaciens
virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the
rpoA
gene encoding the alpha subunit of RNA polymerase is required for the expression of
lacZ
gene under the control of
virB
promoter (
virBp
::
lacZ
) in
E. coli
containing a constitutively active
virG
gene [
virG
(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from
E. coli
and the C-terminal 89 residues from
A. tumefaciens
was able to significantly express
virBp
::
lacZ
in
E. coli
in a VirG(Con)-dependent manner. Utilization of
lac
promoter-driven
virA
and
virG
in combination with the
A. tumefaciens rpoA
construct resulted in significant inducer-mediated expression of the
virBp
::
lacZ
fusion, and the level of
virBp
::
lacZ
expression was positively correlated to the copy number of the
rpoA
construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in
A. tumefaciens
. Furthermore, the effect of sugars on
vir
gene expression was observed only in the presence of the
chvE
gene, suggesting that the glucose-binding protein of
E. coli
, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in
A. tumefaciens
strain A348 from which the
virA
clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the
vir
genes in
E. coli
is less than what is observed in
A. tumefaciens
, suggesting that additional gene(s) from
A. tumefaciens
may be required for the full expression of virulence genes in
E. coli
.
Regulation of
rpoS
Gene Expression in
Pseudomonas
: Involvement of a TetR Family Regulator
Authors: Kojic Milan , Venturi Vittorio
Published: July 2002

The
rpoS
gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase.
rpoS
gene regulation is known to respond to cell density, showing higher expression in stationary phase. For
Pseudomonas aeruginosa,
it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the
rpoS
promoter of
P. putida,
we identified a genomic Tn
5
insertion mutant of
P. putida
which showed a 90% decrease in
rpoS
promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn
5
insertion in a gene, designated
psrA
(
Pseudomonas
sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the
psrA
gene was identified in
P. aeruginosa
; the protein showed 90% identity to PsrA of
P. putida
. A
psrA
::Tn
5
insertion mutant of
P. aeruginosa
was constructed. In both
Pseudomonas
species,
psrA
was genetically linked to the SOS
lexA
repressor gene. Similar to what was observed for
P. putida
, a
psrA
null mutant of
P. aeruginosa
also showed a 90% reduction in
rpoS
promoter activity; both mutants could be complemented for
rpoS
promoter activity when the
psrA
gene was provided in
trans
.
psrA
mutants of both
Pseudomonas
species lost the ability to induce
rpoS
expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate
psrA
gene expression in
Pseudomonas
and in
Escherichia coli
as well as to be capable of activating the
rpoS
promoter in
E. coli
. Our data suggest that PsrA is an important regulatory protein of
Pseudomonas
spp. involved in the regulatory cascade controlling
rpoS
gene regulation in response to cell density.
Analysis of Phase-Specific Gene Expression at the Single-Cell Level in the White-Opaque Switching System of
Candida albicans
Authors: Strauß Anja , Michel Sonja , Morschhäuser Joachim
Published: July 2002

The opportunistic fungal pathogen
Candida albicans
can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous
ura3
mutants from strain WO-1 by targeted gene deletion. The two
URA3
alleles were sequentially inactivated using the
MPA

R

-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using
URA3
as a selection marker. Fluorescence microscopic analysis of cells in which a
GFP
reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific
SAP1
gene was detectably expressed only in opaque cells and that the white-phase-specific
WH11
gene was detectably expressed only in white cells. When
MPA

R

was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the
SAP1
promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when
MPA

R

was placed under the control of the
WH11
promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the
SAP1
and
WH11
genes did not occur at a detectable frequency. The expression of these phase-specific genes of
C. albicans
in vitro, therefore, is tightly linked to the cell type.
Characterization of an Aminoacylase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Story Sherry V. , Grunden Amy M. , Adams Michael W. W.
Published: July 2002

Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon
Pyrococcus furiosus
by its ability to hydrolyze
N
-acetyl-
l
-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl
2
(and to 74% by the addition of CoCl
2
). After reconstitution with ZnCl
2
, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolar
N
-acylated
l
amino acids (Met, Ala, Val, and Leu), as well as
N
-formyl-
l
-methionine. The high

K
m

values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity with
N
-acetyl-
l
-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in the
P. furiosus
genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in
Escherichia coli
by using two approaches. One involved the T7
lac
promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn
2+
ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of the
P. furiosus
aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.
Properties of a Revertant of
Escherichia coli
Viable in the Presence of Spermidine Accumulation: Increase in
l-
Glycerol 3-Phosphate
Authors: Raj V. Samuel , Tomitori Hideyuki , Yoshida Madoka , Apirakaramwong Auayporn , Kashiwagi Keiko , Takio Koji , Ishihama Akira , Igarashi Kazuei
Published: July 2002

Escherichia coli
CAG2242 cells are deficient in the
speG
gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase ς
38
subunit, factors important for cell viability, was increased in the revertant. Levels of
l
-glycerol 3-phosphate also increased in the revertant. Transformation of
glpFK
, which encodes a glycerol diffusion facilitator (
glpF
) and glycerol kinase (
glpK
), to
E. coli
CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that
l
-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that
l
-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.
Developmental Gene Expression in
Bacillus subtilis crsA47
Mutants Reveals Glucose-Activated Control of the Gene for the Minor Sigma Factor ς
H
Authors: Dixon Laurie G. , Seredick Steve , Richer Martin , Spiegelman George B.
Published: July 2002

The presence of excess glucose in growth media prevents normal sporulation of
Bacillus subtilis
. The
crsA47
mutation, located in the gene for the vegetative phase sigma factor (ς
A
) results in a glucose-resistant sporulation phenotype. As part of a study of the mechanisms whereby the mutation in ς
A
overcomes glucose repression of sporulation, we examined the expression of genes involved in sporulation initiation in the
crsA47
background. The
crsA47
mutation had a significant impact on a variety of genes. Changes to stage II gene expression could be linked to alterations in the expression of the
sinI
and
sinR
genes. In addition, there was a dramatic increase in the expression of genes dependent on the minor sigma factor ς
H
. This latter change was paralleled by the pattern of
spo0H
gene transcription in cells with the
crsA47
mutation. In vitro analysis of RNA polymerase containing ς
A47
indicated that it did not have unusually high affinity for the
spo0H
gene promoter. The in vivo pattern of
spo0H
expression is not predicted by the known regulatory constraints on
spo0H
and suggests novel regulation mechanisms that are revealed in the
crsA47
background.
Identification and Characterization of
fhuD1
and
fhuD2
, Two Genes Involved in Iron-Hydroxamate Uptake in
Staphylococcus aureus
Authors: Sebulsky M. Tom , Heinrichs David E.
Published: July 2002

Staphylococcus aureus
can utilize several hydroxamate siderophores for growth under iron-restricted conditions. Previous findings have shown that
S. aureus
possesses a cytoplasmic membrane-associated traffic ATPase that is involved in the specific transport of iron(III)-hydroxamate complexes. In this study, we have identified two additional genes, termed
fhuD1
and
fhuD2
, whose products are involved in this transport process in
S. aureus
. We have shown that
fhuD2
codes for a posttranslationally modified lipoprotein that is anchored in the cytoplasmic membrane, while the deduced amino acid sequence predicts the same for
fhuD1
. The predicted FhuD1 and FhuD2 proteins share 41.0% identity and 56.4% total similarity with each other, 45.9 and 49.1% total similarity with the FhuD homolog in
Bacillus subtilis
, and 29.3 and 24.6% total similarity with the periplasmic FhuD protein from
Escherichia coli
. Insertional inactivation and gene replacement of both genes showed that while FhuD2 is involved in the transport of iron(III) in complex with ferrichrome, ferrioxamine B, aerobactin, and coprogen, FhuD1 shows a more limited substrate range, capable of only iron(III)-ferrichrome and iron(III)-ferrioxamine B transport in
S. aureus
. Nucleotide sequences present upstream of both
fhuD1
and
fhuD2
predict the presence of consensus Fur binding sequences. In agreement, transcription of both genes was negatively regulated by exogenous iron levels through the activity of the
S. aureus
Fur protein.
Escherichia coli
FadR Positively Regulates Transcription of the
fabB
Fatty Acid Biosynthetic Gene
Authors: Campbell John W. , Cronan John E.
Published: July 2002

In
Escherichia coli
expression of the genes of fatty acid degradation (
fad
) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene,
fabA
, is positively regulated by FadR. We report that
fabB
, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and
fadR
-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of
fabB
was regulated in a manner very similar to that of
fabA
expression. A series of genetic and biochemical tests confirmed these observations. Strains containing both
fabB
and
fadR
mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed in
fabA fadR
mutants. A
fadR
strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the
fabB
promoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within the
fabB
promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of
fabB
is positively regulated by FadR.
The Global Regulators GacA and ς
S
Form Part of a Cascade That Controls Alginate Production in
Azotobacter vinelandii
Authors: Castañeda Miguel , Sánchez Judith , Moreno Soledad , Núñez Cinthia , Espı́n Guadalupe
Published: July 2002

Transcription of the
Azotobacter vinelandii algD
gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of
algD
from its three sites during the stationary phase. Here we show that
algD
is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed
A
.
vinelandii
strains that carried mutations in
gacA
encoding the cognate response regulator of GacS and in
rpoS
coding for the stationary-phase ς
S
factor. The
gacA
mutation impaired alginate production and transcription of
algD
from its three promoters. Transcription of
rpoS
was also abolished by the
gacA
mutation. The
rpoS
mutation impaired transcription of
algD
from the p1 promoter and increased it from the p2 ς
E
promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in
A
.
vinelandii
.
Analysis of Transcription of the
Staphylococcus aureus
Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen
Authors: Masalha Mahmud , Borovok Ilya , Schreiber Rachel , Aharonowitz Yair , Cohen Gerald
Published: July 2002

Staphylococcus aureus
is a gram-positive facultative aerobe that can grow in the absence of oxygen by fermentation or by using an alternative electron acceptor. To investigate the mechanism by which
S. aureus
is able to adapt to changes in oxygen concentration, we analyzed the transcriptional regulation of genes that encode the aerobic class Ib and anaerobic class III ribonucleotide reductase (RNR) systems that are responsible for the synthesis of deoxyribonucleotides needed for DNA synthesis. The
S. aureus
class Ib RNR
nrdIEF
and class III RNR
nrdDG
genes and their regulatory regions were cloned and sequenced. Inactivation of the
nrdDG
genes showed that the class III RNR is essential for anaerobic growth. Inhibition of aerobic growth by hydroxyurea showed that the class Ib RNR is an oxygen-dependent enzyme. Northern blot analysis and primer extension analysis demonstrated that transcription of class III
nrdDG
genes is regulated by oxygen concentration and was at least 10-fold higher under anaerobic than under aerobic conditions. In contrast, no significant effect of oxygen concentration was found on the transcription of class Ib
nrdIEF
genes. Disruption or deletion of
S. aureus nrdDG
genes caused up to a fivefold increase in
nrdDG
and
nrdIEF
transcription under anaerobic conditions but not under aerobic conditions. Similarly, hydroxyurea, an inhibitor of the class I RNRs, resulted in increased transcription of class Ib and class III RNR genes under aerobic conditions. These findings establish that transcription of class Ib and class III RNR genes is upregulated under conditions that cause the depletion of deoxyribonucleotide. Promoter analysis of class Ib and class III RNR operons identified several inverted-repeat elements that may account for the transcriptional response of the
nrdIEF
and
nrdDG
genes to oxygen.
Involvement of ResE Phosphatase Activity in Down-Regulation of ResD-Controlled Genes in
Bacillus subtilis
during Aerobic Growth
Authors: Nakano Michiko M. , Zhu Yi
Published: July 2002

The ResD-ResE signal transduction system is required for aerobic and anaerobic respiration in
Bacillus subtilis
. The histidine sensor kinase ResE, by functioning as a kinase and a phosphatase for the cognate response regulator ResD, controls the level of phosphorylated ResD. A high level of phosphorylated ResD is postulated to cause a dramatic increase in transcription of ResDE-controlled genes under anaerobic conditions. A mutant ResE, which retains autophosphorylation and ResD phosphorylation activities but is defective in ResD dephosphorylation, allowed partially derepressed aerobic expression of the ResDE-controlled genes. The result indicates that phosphatase activity of ResE is regulated by oxygen availability and anaerobic induction of the ResDE regulon is partly due to a reduction of the ResE phosphatase activity during anaerobiosis. That elimination of phosphatase activity does not result in complete aerobic derepression suggests that the ResE kinase activity is also subject to control in response to oxygen limitation.
SalmonellaHost Cell Invasion Emerged by Acquisition of a Mosaic of Separate Genetic Elements, IncludingSalmonellaPathogenicity Island 1 (SPI1), SPI5, andsopE2
Authors: Mirold Susanne , Ehrbar Kristin , Weissmüller Astrid , Prager Rita , Tschäpe Helmut , Rüssmann Holger , Hardt Wolf-Dietrich
Published: July 2002

Salmonellaspp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of theSalmonellachromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverseSalmonellaspp., we show here that effector protein genes located in the core of SPI1 are present in allSalmonellalineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of theSalmonellachromosome, namely,sopB(SPI5, centisome 20),sopD(centisome 64), andsopE2(centisomes 40 to 42). Our data demonstrate thatsopB,sopD,andsopE2, along with SPI1, were already present in the last common ancestor of all contemporarySalmonellaspp. Analysis ofSalmonellamutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case ofSalmonella entericaserovar Typhimurium SL1344, by SopE: asopB sopEsopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], andsopE2) and that the last common ancestor of all contemporarySalmonellaspp. was probably already invasive.
Conservative Duplication of Spindle Poles during Meiosis in
Saccharomyces cerevisiae
Authors: Wesp Andreas , Prinz Susanne , Fink Gerald R.
Published: July 2002

During sporulation in diploid
Saccharomyces cerevisiae,
spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that
MPC70/mpc70
heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in
MPC70/mpc70
strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of
MPC70/mpc70
strains lack Mpc70p.
Allelic Diversity and Recombination in
Campylobacter jejuni
Authors: Suerbaum Sebastian , Lohrengel Marc , Sonnevend Agnes , Ruberg Florian , Kist Manfred
Published: July 2002

The allelic diversity and population structure of
Campylobacter jejuni
were studied by multilocus nucleotide sequence analysis. Sequences from seven housekeeping genes were obtained from 32
C. jejuni
isolates isolated from enteritis patients in Germany, Hungary, Thailand, and the United States. Also included was strain NCTC 11168, the complete genomic sequence of which has recently been published. For all loci analyzed, multiple strains carried identical alleles. The frequency of synonymous and nonsynonymous sequence polymorphisms was low. The number of unique alleles per locus ranged from 9 to 15. These alleles occurred in 31 different combinations (sequence types), so that all but two pairs of strains could be distinguished from each other. Sequences were analyzed for evidence of recombination by the homoplasy test and split decomposition. These analyses showed that intraspecific recombination is frequent in
C. jejuni
and has generated extensive diversity of allelic profiles from a small number of polymorphic nucleotides.
Chlorobium tepidum
Mutant Lacking Bacteriochlorophyll
c
Made by Inactivation of the
bchK
Gene, Encoding Bacteriochlorophyll
c
Synthase
Authors: Frigaard Niels-Ulrik , Voigt Ginny D. , Bryant Donald A.
Published: July 2002

The gene encoding bacteriochlorophyll (BChl)
c
synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium
Chlorobium tepidum
and was named
bchK
. The
bchK
mutant of
C. tepidum
was rusty-orange in color and completely lacked BChl
c
. Because of the absence of the BChl
c
antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (<90 μmol m
−2
s
−1
). Various pheophorbides, which probably represent precursors of BChl
c
which had lost magnesium, accumulated in the mutant cells. A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl)
a
and BChl
a
synthases in cells. The amounts of BChl
a
, Chl
a
, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type. This suggests that the BChl
a
antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant. Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes. However, a fraction containing vestigial chlorosomes, denoted “carotenosomes,” was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl
a
species that is probably protein associated. Because of the absence of the strong BChl
c
absorption found in the wild type, the
bchK
mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl
a
in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl
c
and instead used only BChl
a
-containing proteins as the major light-harvesting antennae.
In Vivo DNA-Binding and Oligomerization Properties of the
Shigella flexneri
AraC-Like Transcriptional Regulator VirF as Identified by Random and Site-Specific Mutagenesis
Authors: Porter Megan E. , Dorman Charles J.
Published: July 2002

In
Shigella flexneri
expression of the plasmid-encoded virulence genes is regulated via a complex mechanism involving both environmental signals and specific transactivators. The primary regulator protein, VirF, is a member of the AraC family of transcription factors and shares with other AraC-like proteins a conserved carboxy-terminal domain thought to be important for DNA binding. Random and site-directed mutagenesis of the
virF
gene encoding VirF yielded a number of mutations along the length of the protein which severely affected the ability of VirF to activate gene expression. The mutant proteins were shown to be affected in their ability to activate the virulence genes
virB
and
icsA
, both known to be regulated directly by VirF, as well as the
virB
-dependent virulence gene
mxiC
. Mutating key residues predicted to be important for DNA recognition had a significant negative effect, thereby suggesting that VirF interacts with its target sequence via two helix-turn-helix motifs. Two mutants that were dominant negative when coexpressed with the wild-type VirF protein were also isolated, indicating a role for protein-protein oligomerization in normal VirF function.
A Bacterial Signal Transduction System Controls Genetic Exchange and Motility
Authors: Lang Andrew S. , Beatty J. Thomas
Published: July 2002

The bacterium
Rhodobacter capsulatus
is capable of an unusual form of genetic exchange, mediated by a transducing bacteriophage-like particle called the gene transfer agent (GTA). GTA production by
R. capsulatus
is controlled at the level of transcription by a cellular two-component signal transduction system that includes a putative histidine kinase (CckA) and response regulator (CtrA). We found that, in addition to regulating genetic exchange by
R. capsulatus
, this signal transduction system controls motility. As with the regulation of GTA production, the control of motility by CckA and CtrA occurs through modulation of gene transcription. Disruptions of the
cckA
and
ctrA
genes resulted in a loss of class II, class III, and class IV flagellar gene transcripts, suggesting that
cckA
and
ctrA
function in motility as class I flagellar genes. We also found that, analogous to the GTA genes, transcription of
R. capsulatus
flagellar genes appears to be growth phase dependent: class II flagellar gene transcripts are maximal in the mid-log phase of the culture growth cycle, whereas class III gene transcripts are maximal in the late-log phase of growth. We speculate that coordinate regulation of motility and GTA-mediated genetic exchange in
R. capsulatus
exists because these two processes are complementary mechanisms for cells to cope with unfavorable conditions in natural environments.
Molecular Typing of
Mycobacterium bovis
Isolates from Cameroon
Authors: Njanpop-Lafourcade Berthe M. , Inwald Jacqueline , Ostyn Annick , Durand Benoı̂t , Hughes Steven , Thorel Marie-Françoise , Hewinson Glyn , Haddad Nadia
Published: July 2002

In order to gain a better understanding of the molecular epidemiology of
Mycobacterium bovis
isolates in Cameroon, 75 isolates of
M. bovis
collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with
Dra
I, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS
6110
-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of
M. bovis
strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for
M. bovis
strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.
Automated Ribotyping Provides Rapid Phylogenetic Subgroup Affiliation of Clinical Extraintestinal Pathogenic
Escherichia coli
Strains
Authors: Clermont Olivier , Cordevant Christophe , Bonacorsi Stephane , Marecat Armelle , Lange Marc , Bingen Edouard
Published: July 2002

Using the automated Riboprinter system, we have initiated the construction of an electronic Riboprint database composed of 72 ECOR reference strains and 15 archetypal virulent strains in order to provide a new simple molecular characterization method. More than 90% of the ECOR strains clustered in their original phylogenetic group. All but one of the archetypal virulent strains had a profile identical to that of one of the ECOR strains and could be easily affiliated with a phylogenetic group. This method appears to be an accurate and practical tool especially for investigating the genetic relationship between clinical extraintestinal pathogenic strains and B2 subgroup ECOR strains or archetypal pathotype strains.
Recurrent Soft Tissue Abscesses Caused by
Legionella cincinnatiensis
Authors: Gubler Jacques G. H. , Schorr Mirjam , Gaia V. , Zbinden R. , Altwegg M.
Published: July 2002

Recurrent soft tissue abscesses of the jaw, wrist, and arm developed in a 73-year-old housewife with nephrotic syndrome and immunoglobulin A(κ) gammopathy of unknown etiology. Conventional cultures remained negative, despite visible gram-negative rods on microscopy. Broad-spectrum PCR revealed
Legionella cincinnatiensis
, which was confirmed by isolation of the organism on special
Legionella
medium. Infections due to
Legionella
species outside the lungs are rare.
L. cincinnatiensis
has been implicated in only four cases of clinical infection; these involved the lungs in three patients and the central nervous system in one patient. We conclude that broad-spectrum PCR can be a valuable tool for the evaluation of culture-negative infections with a high probability of bacterial origin and that
Legionella
might be an underdiagnosed cause of pyogenic soft tissue infection.
One Case Each of Recurrent Meningitis and Hemoperitoneum Infection with
Ralstonia mannitolilytica
Authors: Vaneechoutte Mario , De Baere Thierry , Wauters Georges , Steyaert Sophia , Claeys Geert , Vogelaers Dirk , Verschraegen Gerda
Published: July 2002

Two clinical cases of infection with
Ralstonia mannitolilytica
are described: a recurrent meningitis on an implanted intraventricular catheter and an infected hemoperitoneum as a complication of a cholangiocarcinoma. The strains were first misidentified as
Pseudomonas fluorescens
and
Burkholderia cepacia
. Further testing lead to the identification as
Ralstonia pickettii
biovar 3/“
thomasii
,” which was recently shown to represent a separate species,
R. mannitolilytica
(List editor N. Weiss, Int. J. Syst. Evol. Microbiol. 51:795–796, 2001), originally described as
R. mannitolytica
(De Baere et al., Int. J. Syst. Evol. Microbiol. 51:547–558, 2001).
R. mannitolilytica
can be distinguished from all described
Ralstonia
species by its acidification of
d
-arabitol and mannitol and by its lack of nitrate reduction and of alkalinization of tartrate. In order to determine the true prevalence of infections with this species, colistin-resistant “
P. fluorescens
” strains and strains growing on
B. cepacia
selective medium deserve further attention.
Are Two
Cryptococcus neoformans
Strains Epidemiologically Linked?
Authors: Garcia-Hermoso Dea , Dromer Françoise , Mathoulin-Pelissier Simone , Janbon Guilhem
Published: July 2002

The aim of this study was to standardize a method to determine whether two strains of
Cryptococcus neoformans
could be considered epidemiologically linked. We hypothesized that strains isolated from the same patient were epidemiologically linked and that those isolated from different patients were unrelated. We used 17 environmental isolates and 97 clinical isolates from 31 patients diagnosed with cryptococcosis (1 to 14 isolates per patient). Using the plasmid pCnTel-1-labeled probe CENTEL, we were able to differentiate some unrelated strains that yielded the same hybridization profile with the
C. neoformans
middle-repetitive-element CNRE-1 probe. The genetic distances separating the strains isolated from the same patient and those separating the strains isolated from different patients were estimated, and the results obtained with the two probes were compared. Analysis of the results enabled the calculation of two Dice coefficient limits defining the zones containing the pairs of linked strains and the pairs of unrelated strains, as well as an intermediate uncertainty zone for which it was not possible to establish whether the pairs of strains were linked.
Cryptococcus laurentii
Fungemia in a Premature Neonate
Authors: Cheng Ming-Fang , Chiou Christine C. , Liu Yung-Ching , Wang Hao-Zan , Hsieh Kai-Sheng
Published: July 2002

Cryptococcus
spp. other than
Cryptococcus neoformans
are generally considered nonpathogenic to humans. There are only 15 case reports of disease in humans caused by
Cryptococcus laurentii
infection. Underlying diseases and predisposing risk factors seem to play an important role in these cases. Our patient is the first case of an extremely low birth weight infant with
C. laurentii
fungemia reported in the English literature. In our case, the MIC of amphotericin B for
C. laurentii
was 0.25 to 1 μg/ml and the patient had a good outcome following the administration of amphotericin B at 10 mg/kg combined with central venous catheter removal. There will undoubtedly be an increasing occurrence of unusual fungal infections accompanying further advances in medicine. A high degree of suspicion and improvements in the techniques for culture and identification will contribute to the earlier diagnosis and treatment of unusual fungal infections.
Evaluation of Current Methods for Detection of Staphylococci with Reduced Susceptibility to Glycopeptides
Authors: Walsh Timothy R. , Bolmström Anne , Qwärnström Anette , Ho Phion , Wootton Mandy , Howe Robin A. , MacGowan Alasdair P. , Diekema Dan
Published: July 2002

The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant
Staphylococcus aureus
(MRSA) strains and 45
Staphylococcus
strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 μg of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 μg of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was ≥8 mg of vancomycin per liter and ≥8 μg teicoplanin per ml or ≥12 μg of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-)glycopeptide-intermediate
S. aureus
compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (
n
= 48) and MRSA (
n
= 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.
Fluorogenic PCR-Based Quantitative Detection of a Murine Pathogen,
Helicobacter hepaticus
Authors: Ge Zhongming , White Deborah A. , Whary Mark T. , Fox James G.
Published: July 2002

Helicobacter hepaticus
infection in mice is being used as an animal model for elucidating the pathogenesis of gastrointestinal and biliary diseases in humans.
H. hepaticus
, which forms a spreading film on selective agar, is not amenable to routine quantitative counts of organisms in tissues using a CFU method. In this study, a fluorogenic PCR-based assay was developed to quantitatively detect
H. hepaticus
in mouse ceca and feces using the ABI Prism 7700 sequence detection system. A pair of primers and a probe for this assay were generated from the
H. hepaticus cdtB
gene (encoding subunit B of the
H. hepaticus
cytolethal distending toxin). Using this assay, the sensitivity for detection of
H. hepaticus
chromosomal DNA prepared from pure culture was 20 fg, which is equivalent to approximately 14 copies of the
H. hepaticus
genome based on an estimated genome size of ≈1.3 Mb determined by pulsed-field gel electrophoresis.
H. hepaticus
present in feces and cecal samples from
H. hepaticus
-infected mice was readily quantified. The selected PCR primers and probe did not generate fluorescent signals from eight other helicobacters (
H. canis
,
H. cineadi
,
H. felis, H. mustelae, H. nemestrinae, H. pullorum, H. pylori
, and
H. rodentium
)
.
A fluorescent signal was detected from 20 ng of
H. bilis
DNA but with much lower sensitivity (10
6
-fold) than from
H. hepaticus
DNA. Therefore, this assay can be used with high sensitivity and specificity to quantify
H. hepaticus
in experimentally infected mouse models as well as in naturally infected mice.
Genotyping of a Homogeneous Group of
Yersinia pestis
Strains Isolated in the United States
Authors: Huang Xiao-Zhe , Chu May C. , Engelthaler Dave M. , Lindler Luther E.
Published: July 2002

Yersinia pestis
, the causative agent of deadly plague, is considered a reemerging infectious disease and a significant biological terrorism threat. The present project focused on epidemiological investigation of the genetic variability of well-documented strains of
Y. pestis
from the United States by pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis with insertion sequences IS
100
and IS
285
as probes. We examined 37 U.S.
Y. pestis
strains and isolates of a single ribotype, ribotype B, recovered between 1939 and 1998 from patients, animals, and fleas. Our results showed that all isolates had similar PFGE patterns, but minor differences such as missing, additional, and shifted bands were found among almost all strains if they came from different parent strains. The 37 strains and isolates were divided into 26 PFGE types. RFLP analysis with IS
100
as a probe divided these strains and isolates into 16 types, with 43% belonging to IS
100
type 1. Typing with IS
285
as a probe was less specific and led to only four RFLP types, with 81% belonging to type 1. Similarity analysis with BioNumerics software showed that all strains shared ≥80, 86, and 91% similarities on dendrograms prepared from digitized PFGE, IS
100
RFLP analysis, and IS
285
RFLP analysis images, respectively. Our results demonstrate that PFGE offers an increased ability to discriminate between strains (Simpson's index of diversity, 0.98) and therefore can significantly improve epidemiological studies related to the origin of new plague isolates.
Impact of Human Immunodeficiency Virus Type 1 RNA Dimerization on Viral Infectivity and of Stem-Loop B on RNA Dimerization and Reverse Transcription and Dissociation of Dimerization from Packaging
Authors: Shen Ni , Jetté Louis , Liang Chen , Wainberg Mark A. , Laughrea Michael
Published: July 2002

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1
Lai
and HIV-1
Hxb2
), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by ∼50% and proviral DNA synthesis by ∼85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by ∼80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also “nonadditive”; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by ∼50% and viral infectivity by ∼1.4 log. Two mutations, CGCG261→U
UA
A261 (creating a weaker palindrome) and a Δ241–256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.
Interactions between Brain Endothelial Cells and Human T-Cell Leukemia Virus Type 1-Infected Lymphocytes: Mechanisms of Viral Entry into the Central Nervous System
Authors: Romero Ignacio A. , Prevost Marie-Christine , Perret Emmanuelle , Adamson Peter , Greenwood John , Couraud Pierre-Olivier , Ozden Simona
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.