bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial Intestinal Cell Apoptosis Induced byHelicobacter pyloriDepends on Expression of thecagPathogenicity Island Phenotype
Authors: Le'Negrate Gaëlle , Ricci Vittorio , Hofman Véronique , Mograbi Baharia , Hofman Paul , Rossi Bernard
Published: July 2002

Helicobacter pylorihas been shown to induce chronic active gastritis and peptic ulcer and may contribute to the development of duodenal ulcer. Previous studies have shown thatH. pylorimediates apoptosis of gastric epithelial cells via a Fas-dependent pathway. However, evidence for the induction of such a mechanism in intestinal epithelial cells (IEC) byH. pyloriinfection has not been demonstrated yet. This study was performed (i) to ascertain thatH. pylorican induce IEC apoptosis; (ii) to delineate the role of thecagpathogenicity island (PAI),cagE, andvacAgene products in this process; and (iii) to verify whether the Fas-dependent pathway is involved in this phenomenon. When T84 cells were exposed to VacA+/cagPAI+H. pyloristrains (CCUG 17874 and 60190), they exhibited apoptosis hallmarks as assessed by morphological studies, as well as annexin V and 3,3′-dihexyloxacarbocyanine iodide staining. In contrast, few or no apoptotic features could be detected after incubation with an isogenic mutant of strain 60190 in which thecagEgene was disrupted (60190:C−strain) or with a VacA−/cagPAI−H. pyloristrain (G21). In addition, activation of caspase-3 during infection with VacA+/cagPAI+H. pyloristrains was inhibited by pretreatment of IEC with an antagonistic anti-Fas antibody (ZB4). Taken together, these findings indicate thatH. pyloritriggers apoptosis in IEC via a Fas-dependent pathway following a process that depends on the expression of thecagPAI.
Escherichia coli
Hemoglobin Protease Autotransporter Contributes to Synergistic Abscess Formation and Heme-Dependent Growth of
Bacteroides fragilis
Authors: Otto Ben R. , van Dooren Silvy J. M. , Dozois Charles M. , Luirink Joen , Oudega Bauke
Published: July 2002

Intra-abdominal infections (IAI) continue to be a serious clinical problem. Bacterial synergism is an important factor that influences the shift from contamination to IAI, leading to the development of lesions and abscess formation.
Escherichia coli
and
Bacteroides fragilis
are particularly abundant in IAI. The underlying molecular mechanisms of this pathogenic synergy are still unclear. The role of the hemoglobin protease (Hbp) autotransporter protein from
E. coli
in the synergy of IAI was investigated. Hbp is identical to Tsh, a temperature-sensitive hemagglutinin associated with avian pathogenic
E. coli
. Clinical isolates from miscellaneous extraintestinal infections were phenotypically and genotypically screened for Hbp. The presence of Hbp was significantly associated with
E. coli
isolated from IAI and other extraintestinal infections. In a murine infection model, Hbp was shown to contribute to the pathogenic synergy of abscess development. Mice immunized with Hbp were protected against mixed infections and did not develop abscess lesions. Furthermore, an
E. coli
wild-type strain that did not induce abscess formation in the synergy model was transformed with a plasmid encoding the
hbp
gene, and mixed infections with this strain lead to increased growth of
B. fragilis
and induction of abscess lesions. Growth-promoting studies showed that purified Hbp is able to deliver heme to
B. fragilis
strain BE1. In conclusion, results suggest the synergy of abscess formation by
E. coli
and
B. fragilis
can be partly explained by the capacity of
B. fragilis
to intercept Hbp and iron from heme to overcome the iron restrictions imposed by the host.
Expression of Major Surface Protein 2 Variants with Conserved T-Cell Epitopes in
Anaplasma centrale
Vaccinates
Authors: Shkap Varda , Molad Thea , Brayton Kelly A. , Brown Wendy C. , Palmer Guy H.
Published: July 2002

Major surface protein 2 (MSP-2), identified as a protection-inducing immunogen against
Anaplasma marginale
challenge, is an immunodominant outer membrane protein with orthologues in all examined
Anaplasma
species. Although immunization with live
Anaplasma centrale
has long been used to induce protection against acute disease upon challenge with virulent
A. marginale
, its MSP-2 structure and whether MSP-2 variants are generated during persistence of the vaccine strain was unknown. In this study, we showed that the
A. centrale
vaccine strain persisted for a minimum of 4 years postvaccination and generated sequential MSP-2 variants. Comparison of amino acid sequences encoded by
A. centrale msp-2
transcripts from the initial postimmunization period and from sequential time points during persistence of the vaccine strain revealed a central hypervariable domain flanked by conserved amino and carboxy-terminal regions. This structure corresponded to that shown in
A. marginale
MSP-2, where the central hypervariable region encodes variant B-cell epitopes in the extracellular domain and the flanking transmembrane domains are rich in CD4
+
-T-cell epitopes. Importantly, at least four CD4
+
-T-cell epitopes are conserved between the two species, a finding consistent with
A. marginale
challenge triggering a recall response of CD4
+
T cells induced by
A. centrale
vaccination. The genomic arrangement is conserved between
A. centrale
and
A. marginale
with multiple
msp-2
pseudogenes and a single operon-linked expression site for the full-length
msp-2
. This conservation of both genomic structure for generating MSP-2 variants and the CD4
+
-T-cell epitopes between these two genetically distinct
Anaplasma
species indicates that they present a similar repertoire of MSP-2 epitopes to the immune system and that this similarity may be responsible for all or part of the
A. centrale
vaccine efficacy.
Inactivation of the
Moraxella catarrhalis
Superoxide Dismutase SodA Induces Constitutive Expression of Iron-Repressible Outer Membrane Proteins
Authors: Luke Nicole R. , Karalus Richard J. , Campagnari Anthony A.
Published: July 2002

Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen
Moraxella catarrhalis
, and the gene responsible for the SOD activity,
sodA
, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned
M. catarrhalis
7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic
M. catarrhalis sodA
mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of
E. coli
QC774, a
sodA sodB
-deficient mutant, demonstrated the functional expression of SOD activity by cloned
M. catarrhalis sodA
. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that
M. catarrhalis
SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that
M. catarrhalis
strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate.
Cortactin Is Necessary for F-Actin Accumulation in Pedestal Structures Induced by Enteropathogenic
Escherichia coli
Infection
Authors: Cantarelli Vlademir V. , Takahashi Akira , Yanagihara Itaru , Akeda Yukihiro , Imura Kinichi , Kodama Toshio , Kono Gengo , Sato Yoshihisa , Iida Tetsuya , Honda Takeshi
Published: July 2002

Cortactin and the translocated intimin receptor, Tir, interacted with each other in pedestal formation in HeLa cells infected with enteropathogenic
Escherichia coli
(EPEC). Cortactin is shown to be necessary for organizing actin pedestals in response to EPEC, based on the expression of green fluorescent protein-fused cortactin derivatives in HeLa cells.
Gel Shift Assay of Nuclear Extracts from
Histoplasma capsulatum
Demonstrates the Presence of Several DNA Binding Proteins
Authors: Ignatov Atanas , Keath Elizabeth J.
Published: July 2002

A gel shift assay was optimized to detect several general DNA binding proteins from
Histoplasma capsulatum
strain G217B. The electrophoretic mobility shift assay (EMSA) technique also detected protein(s) recognizing a pyrimidine-rich motif found in several
Histoplasma
promoters. Establishment of EMSA conditions provides an important framework to evaluate regulation of homeostatic or phase-specific genes that may influence virulence in
Histoplasma
and other dimorphic fungal pathogens.
A DNA Vaccine Encoding Lumazine Synthase fromBrucella abortusInduces Protective Immunity in BALB/c Mice
Authors: Velikovsky Carlos A. , Cassataro Juliana , Giambartolomei Guillermo H. , Goldbaum Fernando A. , Estein Silvia , Bowden Raul A. , Bruno Laura , Fossati Carlos A. , Spitz Moisés
Published: July 2002

This study was conducted to evaluate the immunogenicity of theBrucella abortuslumazine synthase (BLS) gene cloned into the pcDNA3 plasmid, which is driven by the cytomegalovirus promoter. Injection of plasmid DNA carrying the BLS gene (pcDNA-BLS) into BALB/c mice elicited both humoral and cellular immune responses. Antibodies to the encoded BLS included immunoglobulin G1 (IgG1) IgG2a, IgG2b, IgG3, and IgM isotypes. Animals injected with pcDNA-BLS exhibited a dominance of IgG2a over IgG1. In addition, spleen cells from vaccinated animals produced interleukin-2 and gamma interferon but not IL-10 or IL-4 after in vitro stimulation with recombinant BLS (rBLS), suggesting the induction of a Th1 response. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged withB. abortus544. Immunization with pcDNA-BLS- reduced the bacterial burden relative to those in the control groups. Mice immunized with rBLS produced a significant humoral response but did not show a specific cellular response or any protection from challenge. Altogether, these data suggest that pcDNA-BLS is a good immunogen for the production of humoral and cell-mediated responses in mice and is a candidate for use in future studies of vaccination against brucellosis.
Differential Persistence among Genomovars of theBurkholderia cepaciaComplex in a Murine Model of Pulmonary Infection
Authors: Chu Karen K. , Davidson Donald J. , Halsey T. Keith , Chung Jacqueline W. , Speert David P.
Published: July 2002

Cystic fibrosis patients infected with strains from different genomovars of theBurkholderia cepaciacomplex can experience diverse clinical outcomes. To identify genomovar-specific determinants that might be responsible for these differences, we developed a pulmonary model of infection in BALB/c mice. Mice were rendered leukopenic by administration of cyclophosphamide prior to intranasal challenge with 1.6 × 104bacteria. Five of six genomovar II strains persisted at stable numbers in the lungs until day 16 with minimal toxicity, whereas zero of seven genomovar III strains persisted but resulted in variable toxicity. We have developed a chronic pulmonary model ofB. cepaciainfection which reveals differences among genomovars in terms of clinical infection outcome.
Borrelia burgdorferi
Population Kinetics and Selected Gene Expression at the Host-Vector Interface
Authors: Hodzic Emir , Feng Sunlian , Freet Kimberly J. , Borjesson Dori L. , Barthold Stephen W.
Published: July 2002

By using real-time quantitative PCR, the population dynamics and gene transcription of
Borrelia burgdorferi
were examined in ticks and skin of mice during acquisition of the infection from mice by ticks and during transmission of the infection from ticks to mice. Population dynamics were determined by using a
flaB
DNA target. A quantitative analysis of
flaB
,
ospA
,
ospC
,
dbpA
, and
arp
transcription was also performed. The results revealed that both uninfected larval and nymphal
Ixodes scapularis
ticks acquired
B. burgdorferi
as early as 1 day after attachment and that the sizes of spirochete populations within ticks increased during feeding. In addition, all gene targets revealed that there was RNA transcription during feeding. Similar events occurred within infected nymphal ticks feeding on uninfected hosts. Transmission from infected nymphal ticks to mice could be detected within 1 day after attachment. Analysis of skin during the first 3 days after attachment of infected ticks revealed rising numbers of spirochetes but minimal gene transcription. In contrast, the skin of mice with established infections revealed static populations of spirochetes and active but stable transcription of
flaB
,
ospC
,
dbpA
, and
arp
. There were consistent reductions in the number of spirochetes in the skin at the tick attachment sites compared to the number of spirochetes in the skin at nontick sites, but there were no differences in gene expression between tick and nontick skin sites. Evidence of
ospA
transcription in skin could be found 1 day after tick attachment but not thereafter.
Characterization of
Salmonella enterica
Derivatives Harboring Defined
aroC
and
Salmonella
Pathogenicity Island 2 Type III Secretion System (
ssaV
) Mutations by Immunization of Healthy Volunteers
Authors: Hindle Zoë , Chatfield Steven N. , Phillimore Jo , Bentley Matthew , Johnson Julie , Cosgrove Catherine A. , Ghaem-Maghami Marjan , Sexton Amy , Khan Mohammad , Brennan Frank R. , Everest Paul , Wu Tao , Pickard Derek , Holden David W. , Dougan Gordon , Griffin George E. , House Deborah , Santangelo Joseph D. , Khan Shahid A. , Shea Jaqueline E. , Feldman Robert G. , Lewis David J. M.
Published: July 2002

The attenuation and immunogenicity of two novel
Salmonella
vaccine strains,
Salmonella enterica
serovar Typhi (Ty2 Δ
aroC
Δ
ssaV
, designated ZH9) and
S. enterica
serovar Typhimurium (TML Δ
aroC
Δ
ssaV
, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10
7
, 10
8
, or 10
9
CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10
8
and two of three receiving 10
9
CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10
8
and 10
9
CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10
9
CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.
Color-Sensitive Motility and Methanol Release Responses in
Rhodobacter sphaeroides
Authors: Kort Remco , Crielaard Wim , Spudich John L. , Hellingwerf Klaas J.
Published: July 2002

Blue-light-induced repellent and demethylation responses, characteristic of behavioral adaptation, were observed in
Rhodobacter sphaeroides
. They were analyzed by computer-assisted motion analysis and through the release of volatile tritiated compounds from [
methyl
-
3
H]methionine-labeled cells, respectively. Increases in the stop frequency and the rate of methanol release were induced by exposure of cells to repellent light signals, such as an increase in blue- and a decrease in infrared-light intensity. At a λ of >500 nm the amplitude of the methanol release response followed the absorbance spectrum of the photosynthetic pigments, suggesting that they function as photosensors for this response. In contrast to the previously reported motility response to a decrease in infrared light, the blue-light response reported here does not depend on the number of photosynthetic pigments per cell, suggesting that it is mediated by a separate sensor. Therefore, color discrimination in taxis responses in
R. sphaeroides
involves two photosensing systems: the photosynthetic pigments and an additional photosensor, responding to blue light. The signal generated by the former system could result in the migration of cells to a light climate beneficial for photosynthesis, while the blue-light system could allow cells to avoid too-high intensities of (harmful) blue light.
Vibrio vulnificus
Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin Gene
vvhA
Authors: Lee Shee Eun , Shin Sung Heui , Kim Soo Young , Kim Young Ran , Shin Dong Hyeon , Chung Sun Sik , Lee Zang Hee , Lee Jee Yeon , Jeong Kwang Choel , Choi Sang Ho , Rhee Joon Haeng
Published: July 2002

In an attempt to dissect the virulence regulatory mechanism in
Vibrio vulnificus
, we tried to identify the
V. cholerae
transmembrane virulence regulator
toxRS
(
toxRS
Vc
) homologs in
V. vulnificus
. By comparing the sequences of
toxRS
of
V. cholerae
and
V. parahaemolyticus
(
toxRS
Vp
), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb
Bgl
II-
Hin
dIII fragment and a 1.2-kb
Hin
dIII fragment containing two complete open reading frames and one partial open reading frame attributable to
toxR
Vv
,
toxS
Vv
, and
htpG
Vv
were cloned. ToxR
Vv
shared 55.0 and 63.0% sequence homology with ToxR
Vc
and ToxR
Vp
, respectively. ToxS
Vv
was 71.5 and 65.7% homologous to ToxS
Vc
and ToxS
Vp
, respectively. The amino acid sequences of ToxRS
Vv
showed transmembrane and activity domains similar to those observed in ToxRS
Vc
and ToxRS
Vp
. Western blot analysis proved the expression of ToxR
Vv
in
V. vulnificus
. ToxRS
Vv
enhanced, in an
Escherichia coli
background, the expression of the
V. vulnificus
hemolysin gene (
vvhA
) fivefold. ToxRS
Vv
also activated the ToxR
Vc
-regulated
ctx
promoter incorporated into an
E. coli
chromosome. A
toxR
Vv
null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The
toxR
Vv
mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR
Vv
may regulate the virulence expression of
V. vulnificus
.
Alfalfa Root Nodule Invasion Efficiency Is Dependent on
Sinorhizobium meliloti
Polysaccharides
Authors: Pellock Brett J. , Cheng Hai-Ping , Walker Graham C.
Published: July 2002

The soil bacterium
Sinorhizobium meliloti
is capable of entering into a nitrogen-fixing symbiosis with
Medicago sativa
(alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by
S. meliloti
are crucial for establishing this symbiosis. Alfalfa nodule invasion by
S. meliloti
can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled
S. meliloti
cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how
S. meliloti
polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.
Global Gene Expression Profiles of
Bacillus subtilis
Grown under Anaerobic Conditions
Authors: Ye Rick W. , Tao Wang , Bedzyk Laura , Young Thomas , Chen Mario , Li Liao
Published: July 2002

Bacillus subtilis
can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.
Vibrio fischeri lux
Genes Play an Important Role in Colonization and Development of the Host Light Organ
Authors: Visick Karen L. , Foster Jamie , Doino Judith , McFall-Ngai Margaret , Ruby Edward G.
Published: July 2002

The bioluminescent bacterium
Vibrio fischeri
and juveniles of the squid
Euprymna scolopes
specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (
lux
) genes of symbiotic
V. fischeri
have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (
luxA
,
luxI
, and
luxR
), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these
lux
mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in
trans
, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of
lux
mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of
V. fischeri
genes that affect normal host tissue development.
A Family of Six Flagellin Genes Contributes to the
Caulobacter crescentus
Flagellar Filament
Authors: Ely Bert , Ely Tracey W. , Crymes William B. , Minnich Scott A.
Published: July 2002

The
Caulobacter crescentus
flagellar filament is assembled from multiple flagellin proteins that are encoded by six genes. The amino acid sequences of the FljJ and FljL flagellins are divergent from those of the other four flagellins. Since these flagellins are the first to be assembled in the flagellar filament, one or both might have specialized to facilitate the initiation of filament assembly.
Reduction of GC → TA Transversion Mutation by Overexpression of MutS in
Escherichia coli
K-12
Authors: Zhao Jingyong , Winkler Malcolm E.
Published: July 2002

Overexpression of the MutS repair protein significantly decreased the rate of
lacZ
GC → TA transversion mutation in stationary-phase and exponentially growing bacteria and in
mutY
and
mutM
mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC → TA transversion increased in
mutL
or
mutS
mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce
lacZ
AT → CG transversion mutation in a
mutT
mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in
Escherichia coli
cells but may not be able to compete with mutation fixation by MutY in
mutT
mutants.
Aerotolerance and Peroxide Resistance in Peroxidase and PerR Mutants of
Streptococcus pyogenes
Authors: King Katherine Y. , Horenstein Joshua A. , Caparon Michael G.
Published: July 2002

Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism
Streptococcus pyogenes
, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (
ahpC
) and glutathione peroxidase (
gpoA
). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that
S. pyogenes
demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in
ahpC
and
gpoA
mutants but not in mutants lacking PerR, a repressor of several genes including
ahpC
and catalase (
katA
) in
Bacillus subtilis
. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in
S. pyogenes
, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.
HbpR, a New Member of the XylR/DmpR Subclass within the NtrC Family of Bacterial Transcriptional Activators, Regulates Expression of 2-Hydroxybiphenyl Metabolism in
Pseudomonas azelaica
HBP1
Authors: Jaspers Marco C. M. , Suske Winfried A. , Schmid Andreas , Goslings David A. M. , Kohler Hans-Peter E. , van der Meer Jan Roelof
Published: July 2002

The regulation of 2-hydroxybiphenyl and 2,2′-dihydroxybiphenyl degradation in
Pseudomonas azelaica
is mediated by the regulatory gene,
hbpR
. The
hbpR
gene encodes a 63-kDa protein belonging to the NtrC family of prokaryotic transcriptional activators and having the highest homology to members of the XylR/DmpR subclass. Disruption of the
hbpR
gene in
P. azelaica
and complementation in
trans
showed that the HbpR protein was the key regulator for 2-hydroxybiphenyl metabolism. Induction experiments with
P. azelaica
and
Escherichia coli
containing
luxAB
-based transcriptional fusions revealed that HbpR activates transcription from a promoter (P

hbpC

) in front of the first gene for 2-hydroxybiphenyl degradation,
hbpC
, and that 2-hydroxybiphenyl itself is the direct effector for HbpR-mediated activation. Of several compounds tested, only the pathway substrates 2-hydroxybiphenyl and 2,2′-dihydroxybiphenyl and structural analogs like 2-aminobiphenyl and 2-hydroxybiphenylmethane were effectors for HbpR activation. HbpR is therefore, to our knowledge, the first regulator of the XylR/DmpR class that recognizes biaromatic but not monoaromatic structures. Analysis of a spontaneously occurring mutant,
P. azelaica
HBP1 Prp, which can grow with the non-wild-type effector 2-propylphenol, revealed a single mutation in the
hbpR
gene (T613C) leading to a Trp→Arg substitution at amino acid residue 205.
P. azelaica
HBP1 derivative strains without a functional
hbpR
gene constitutively expressed the genes for 2-hydroxybiphenyl degradation when complemented in
trans
with the
hbpR
-T613C gene. This suggests the importance of this residue, which is conserved among all members of the XylR/DmpR subclass, for interdomain repression.
Identification and Characterization of a New Prespore-Specific Regulatory Gene,
rsfA
, of
Bacillus subtilis
Authors: Juan Wu Ling , Errington Jeff
Published: July 2002

Differential gene expression during
Bacillus subtilis
sporulation is controlled by sigma factors and other regulatory effectors. The first compartmentalized sigma factor, ς
F
, is active specifically in the prespore compartment. During our screening for new chromosome segregation mutants using a ς
F
-dependent
gpr-lacZ
reporter as a probe, we identified a new gene (
ywfN
) required for maximal expression of the reporter and named it
rsfA
. The product of
rsfA
has features of gene regulatory proteins, and the protein colocalizes with DNA. The expression of
rsfA
is under the control of both ς
F
and ς
G
. Null mutations in
rsfA
have different effects on the expression of ς
F
-dependent genes, suggesting that the RsfA protein is a regulator of transcription that fine-tunes gene expression in the prespore.
Hydrogen Peroxide Activates the SoxRS Regulon In Vivo
Authors: Manchado Manuel , Michán Carmen , Pueyo Carmen
Published: July 2002

By multiplex reverse transcription-PCR, we demonstrate that the SoxRS response, which protects cells against superoxide toxicity, is triggered also by hydrogen peroxide. SoxR-dependent inductions of 7.3-, 7.6-, 4.6-, 2.2-, and 2.6-fold were quantified for
soxS
,
micF
,
sodA
,
inaA
, and
fpr
transcripts, respectively. This finding suggests an extensive and tight connectivity between different regulatory pathways in the
Escherichia coli
response to oxidative stress.
Membrane Redistribution of the
Escherichia coli
MinD Protein Induced by MinE
Authors: Rowland S. L. , Fu X. , Sayed M. A. , Zhang Y. , Cook W. R. , Rothfield L. I.
Published: July 2002

Escherichia coli
cells contain potential division sites at midcell and adjacent to the cell poles. Selection of the correct division site at midcell is controlled by three proteins: MinC, MinD, and MinE. It has previously been shown (D. Raskin and P. de Boer, Cell 91:685–694, 1997) that MinE-Gfp localizes to the midcell site in an MinD-dependent manner. We use here Gfp-MinD to show that MinD associates with the membrane around the entire periphery of the cell in the absence of the other Min proteins and that MinE is capable of altering the membrane distribution pattern of Gfp-MinD. Studies with the isolated N-terminal and C-terminal MinE domains indicated different roles for the two MinE domains in the redistribution of membrane-associated MinD.
Alteration of the Lipopolysaccharide Structure Affects the Functioning of the Xcp Secretory System in
Pseudomonas aeruginosa
Authors: Michel Gérard , Ball Geneviève , Goldberg Joanna B. , Lazdunski Andrée
Published: July 2002

Pseudomonas aeruginosa
secretes a wide range of hydrolytic enzymes into the external medium by the Xcp secretion machinery. To better understand the role played by envelope constituents in the functioning of this type II secretory system, we have studied the influence of lipopolysaccharide (LPS) on the secretion of two extracellular enzymes, the elastase LasB and the lipase LipA. Strains with defective LPS decreased production of LasB and altered the secretion processes of both LasB and LipA without any apparent effect on the composition of the Xcp machinery. The PAO1
algC
strain, defective in the outer core of LPS, was leaky, as shown by the extracellular release of the periplasmic β-lactamase. Generation of an
xcpR
mutation in this mutant led only to a partial accumulation of LasB within the cells, indicating that in strain PAO1
algC
with a functional
xcpR
gene, LasB was released in the extracellular medium partly by leakage and partly by secretion. The pool of LasB released into the medium by leakage was not recovered in an active form, while extracellular LasB was active when secreted via the secretory machinery. Further analysis revealed that the presence of a functional Xcp machinery is strictly required for the activation process of LasB. Our results provide evidence that the Xcp system is not fully functional when the LPS structure of
P. aeruginosa
is altered.
Functional Domains of the TOL Plasmid Transcription Factor XylS
Authors: Kaldalu Niilo , Toots Urve , de Lorenzo Victor , Ustav Mart
Published: July 2002

The alkylbenzoate degradation genes of
Pseudomonas putida
TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the
Pm
operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the
Pm
promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.
Genetic and Biochemical Characterization of
Salmonella enterica
Serovar Typhi Deoxyribokinase
Authors: Tourneux Lise , Bucurenci Nadia , Saveanu Cosmin , Kaminski Pierre Alexandre , Bouzon Madeleine , Pistotnik Elisabeth , Namane Abdelkader , Marlière Philippe , Bârzu Octavian , Li de la Sierra Inès , Neuhard Jan , Gilles Anne-Marie
Published: July 2002

We identified in the genome of
Salmonella enterica
serovar Typhi the gene encoding deoxyribokinase,
deoK
. Two other genes, vicinal to
deoK
, were determined to encode the putative deoxyribose transporter (
deoP
) and a repressor protein (
deoQ
). This locus, located between the
uhpA
and
ilvN
genes, is absent in
Escherichia coli
. The
deoK
gene inserted on a plasmid provides a selectable marker in
E. coli
for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi,
S. enterica
serovar Typhimurium, or
E. coli
. The catalytic properties of the recombinant deoxyribokinase overproduced in
E. coli
correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and
E. coli
ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the
V
max
of deoxyribokinase by a factor of 2.5 and increased the
K
m
for deoxyribose by a factor of 70, compared to the parent enzyme.
Substrate Range and Genetic Analysis of
Acinetobacter
Vanillate Demethylase
Authors: Morawski Birgit , Segura Ana , Ornston L. Nicholas
Published: July 2002

An
Acinetobacter
sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs
m
-anisate,
m
-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type
Acinetobacter
bacteria. PCR mutagenesis of
vanAB
, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.
Identification of the Active Site of HetR Protease and Its Requirement for Heterocyst Differentiation in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Dong Yuqing , Huang Xu , Wu Xiang-Yu , Zhao Jindong
Published: July 2002

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from
Anabaena
sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant
hetR
gene was introduced into
Anabaena
strain 884a, a
hetR
mutant. The resultant strain,
Anabaena
strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant
hetR
gene was induced upon removal of combined nitrogen. The
Anabaena
strain 216, which carries a mutant
hetR
gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.
Activation of SoxR by Overproduction of Desulfoferrodoxin: Multiple Ways To Induce the
soxRS
Regulon
Authors: Gaudu Philippe , Dubrac Sarah , Touati Danièle
Published: July 2002

The
soxRS
response, which protects cells against superoxide toxicity, is triggered by the oxidation of SoxR, a transcription factor. Superoxide excess and NADPH depletion induce the regulon. Unexpectedly, we found that the overproduction of desulfoferrodoxin, a superoxide reductase from sulfate-reducing bacteria, also induced this response. We suggest that desulfoferrodoxin interferes with the reducing pathway that keeps SoxR in its inactive form.
Mobilization of Plasmids and Chromosomal DNA Mediated by the SXT Element, a Constin Found in
Vibrio cholerae
O139
Authors: Hochhut Bianca , Marrero Joeli , Waldor Matthew K.
Published: July 2002

The
Vibrio cholerae
SXT element encodes resistance to multiple antibiotics and is a conjugative, self-transmissible, and chromosomally integrating element (a constin). Excision and self-transfer of the SXT element require an element-encoded integrase. We now report that the SXT element can also mobilize the plasmids RSF1010 and CloDF13 in
trans
as well as chromosomal DNA in an Hfr-like manner. SXT element-mediated mobilization of plasmids and chromosomal DNA, unlike its self-transfer, is not dependent upon excision of the element from the chromosome. These results raise the possibility that the SXT element and other constins play a general role in horizontal gene transfer among gram-negative bacteria.
RegG, a CcpA Homolog, Participates in Regulation of Amylase-Binding Protein A Gene (
abpA
) Expression in
Streptococcus gordonii
Authors: Rogers Jeffrey D. , Scannapieco Frank A.
Published: July 2002

The amylase-binding protein A (AbpA) of
Streptococcus gordonii
was found to be undetectable in supernatants of mid-log-phase cultures containing >1% glucose but abundant in supernatants of cultures made with brain heart infusion (BHI), which contains 0.2% glucose. A 10-fold decrease in the level of
abpA
mRNA in
S. gordonii
cells cultured in BHI was noted after the addition of glucose to 1%. Analysis of the
abpA
sequence revealed a potential catabolite responsive element CRE 153 bp downstream of the putative translational start site. A catabolite control protein A gene (
ccpA
) homolog from
S. gordonii
, designated
regG
, was cloned. A
regG
mutant strain demonstrated moderately less repression of
abpA
transcription in the presence of 1% glucose. Diauxic growth with glucose and lactose was not affected in the RegG mutant compared to the wild-type parental strain. These results suggest that while RegG plays a role in
abpA
expression, other mechanisms of catabolite repression are present.
Anatomical Analysis of
Saccharomyces cerevisiae
Stalk-Like Structures Reveals Spatial Organization and Cell Specialization
Authors: Scherz Ruth , Shinder Vera , Engelberg David
Published: July 2002

Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures. These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter). They are formed in response to UV radiation of cultures spread on high agar concentrations. Here we present an anatomical analysis of the stalks. Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core. This outer layer is physically separated from the core and contains many dead cells. The outer layer may form a protective shell for the core cells. Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells. We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization. We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology. Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures. Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects. The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.
Cytochrome Complex Essential for Photosynthetic Oxidation of both Thiosulfate and Sulfide in
Rhodovulum sulfidophilum
Authors: Appia-Ayme Corinne , Little Phillip J. , Matsumoto Yumi , Leech Andrew P. , Berks Ben C.
Published: July 2002

Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation. A thiosulfate-induced cytochrome
c
has been purified from the photosynthetic α-proteobacterium
Rhodovulum sulfidophilum
. This cytochrome
c
551
is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit. The cytochrome
c
551
structural genes are part of an 11-gene
sox
locus. Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination. A
soxA
mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide. Cytochrome
c
551
is thus essential for the metabolism of both these sulfur species. Periplasmic extracts of wild-type
R. sulfidophilum
exhibit thiosulfate:cytochrome
c
oxidoreductase activity. However, such activity can only be measured for a
soxA
mutant strain if the periplasmic extract is supplemented with purified cytochrome c
551
. Gene clusters similar to the
R. sulfidophilum sox
locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.
Roles of Glutamate Synthase,gltBD, andgltFin Nitrogen Metabolism ofEscherichia coliandKlebsiella aerogenes
Authors: Goss Thomas J. , Perez-Matos Ana , Bender Robert A.
Published: July 2002

Mutants ofEscherichia coliandKlebsiella aerogenesthat are deficient in glutamate synthase (glutamate-oxoglutarate amidotransferase [GOGAT]) activity have difficulty growing with nitrogen sources other than ammonia. Two models have been proposed to account for this inability to grow. One model postulated an imbalance between glutamine synthesis and glutamine degradation that led to a repression of the Ntr system and the subsequent failure to activate transcription of genes required for the use of alternative nitrogen sources. The other model postulated that mutations ingltBorgltD(which encode the subunits of GOGAT) were polar on a downstream gene,gltF, which is necessary for proper activation of gene expression by the Ntr system. The data reported here show that thegltFmodel is incorrect for three reasons: first, a nonpolargltBand a polargltDmutation ofK. aerogenesboth show the same phenotype; second,K. aerogenesand several other enteric bacteria lack a gene homologous togltF; and third, mutants ofE. coliwhosegltFgene has been deleted show no defect in nitrogen metabolism. The argument that accumulated glutamine represses the Ntr system ingltBorgltDmutants is also incorrect, because these mutants can derepress the Ntr system normally so long as sufficient glutamate is supplied. Thus, we conclude thatgltBorgltDmutants grow slowly on many poor nitrogen sources because they are starved for glutamate. Much of the glutamate formed by catabolism of alternative nitrogen sources is converted to glutamine, which cannot be efficiently converted to glutamate in the absence of GOGAT activity. Finally, GOGAT-deficientE. colicells growing with glutamine as the sole nitrogen source increase their synthesis of the other glutamate-forming enzyme, glutamate dehydrogenase, severalfold, but this is still insufficient to allow rapid growth under these conditions.