bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of OspE-Related, OspF-Related, and Elp Lipoproteins of
Borrelia burgdorferi
Strain 297 by Mammalian Host-Specific Signals
Authors: Hefty P. Scott , Jolliff Sarah E. , Caimano Melissa J. , Wikel Stephen K. , Radolf Justin D. , Akins Darrin R.
Published: July 2002

In previous studies we have characterized the cp32/18 loci in
Borrelia burgdorferi
297 which encode OspE and OspF orthologs and a third group of lipoproteins which possess OspE/F-like leader peptides (Elps). To further these studies, we have comprehensively analyzed their patterns of expression throughout the borrelial enzootic cycle. Serial dilution reverse transcription-PCR analysis indicated that although a shift in temperature from 23 to 37°C induced transcription for all nine genes analyzed, this effect was often markedly enhanced in mammalian host-adapted organisms cultivated within dialysis membrane chambers (DMCs) implanted within the peritoneal cavities of rats. Indirect immunofluorescence assays performed on temperature-shifted, in vitro-cultivated spirochetes and organisms in the midguts of unfed and fed ticks revealed distinct expression profiles for many of the OspE-related, OspF-related, and Elp proteins. Other than BbK2.10 and ElpA1, all were expressed by temperature-shifted organisms, while only OspE, ElpB1, OspF, and BbK2.11 were expressed in the midguts of fed ticks. Additionally, although mRNA was detected for all nine lipoprotein-encoding genes, two of these proteins (BbK2.10 and ElpA1) were not expressed by spirochetes cultivated in vitro, within DMCs, or by spirochetes within tick midguts. However, the observation that
B. burgdorferi
-infected mice generated specific antibodies against BbK2.10 and ElpA1 indicated that these antigens are expressed only in the mammalian host and that a form of posttranscriptional regulation is involved. Analysis of the upstream regions of these genes revealed several differences between their promoter regions, the majority of which were found in the −10 and −35 hexamers and the spacer regions between them. Also, rather than undergoing simultaneous upregulation during tick feeding, these genes and the corresponding lipoproteins appear to be subject to progressive recruitment or enhancement of expression as
B. burgdorferi
is transmitted from its tick vector to the mammalian host. These findings underscore the potential relevance of these molecules to the pathogenic events of early Lyme disease.
Endogenous Interleukin-10 Is Required for Prevention of a Hyperinflammatory Intracerebral Immune Response inListeria monocytogenesMeningoencephalitis
Authors: Deckert Martina , Soltek Sabine , Geginat Gernot , Lütjen Sonja , Montesinos-Rongen Manuel , Hof Herbert , Schlüter Dirk
Published: July 2002

To analyze the role of interleukin-10 (IL-10) in bacterial cerebral infections, we studied cerebral listeriosis in IL-10-deficient (IL-10−/−) and wild-type (WT) mice, the latter of which express high levels of IL-10 in both primary and secondary cerebral listeriosis. IL-10−/−mice succumbed to primary as well as secondary listeriosis, whereas WT mice were significantly protected from secondary listeriosis by prior intraperitoneal immunization withListeria monocytogenes. Meningoencephalitis developed in both strains; however, in IL-10−/−mice the inflammation was more severe and associated with increased brain edema and multiple intracerebral hemorrhages. IL-10−/−mice recruited significantly increased numbers of leukocytes, in particular granulocytes, to the brain, and the intracerebral cytokine (tumor necrosis factor, IL-1, IL-12, gamma interferon, and inducible nitric oxide synthase) and chemokine (crg2/IP-10, RANTES, MuMig, macrophage inflammatory protein 1α [MIP-1α], and MIP-1β) transcription was enhanced compared to that in WT mice. Despite this prominent hyperinflammation, the frequencies of intracerebralL. monocytogenes-specific CD8+T cells were reduced and the intracerebral bacterial load was not reduced in IL-10−/−mice compared to WT mice. Following intraperitoneal infection, IL-10−/−mice exhibited hepatic hyperinflammation without better bacterial clearance; however, in contrast to the mice with cerebral listeriosis, they did not succumb, illustrating that intrinsic factors of the target organ have a strong impact on the course and outcome of the infection.
Proteolytic Inhibition ofSalmonella entericaSerovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells
Authors: Mynott Tracey L. , Crossett Ben , Prathalingam S. Radhika
Published: July 2002

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenicEscherichia coliinfection. In this study, we examined the effect of bromelain onSalmonella entericaserovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.
Listeria monocytogenesInfection in Caspase-11-Deficient Mice
Authors: Mueller Nicolas J. , Wilkinson Robert A. , Fishman Jay A.
Published: July 2002

Caspase-11 (Cas11) is a cysteine protease involved in programmed cell death and cytokine maturation. Through activation of Cas1 (interleukin-1β [IL-1β]-converting enzyme), Cas11 is directly involved in the maturation of IL-1β and IL-18. Apoptosis is mediated through Cas3. Given the role of apoptosis and cytokine signaling during the innate immune response in intracellular infection, we examined Cas11-deficient (Cas11−/−) mice during infection withListeria monocytogenes. Cas11−/−and wild-type C57BL/6 mice were equally susceptible to intravenous infection withL. monocytogenes, resulting in similar bacterial burdens in tissue and similar survival rates. By contrast, enhanced susceptibility was observed in control mice on a mixed genetic 129/C57BL/DBA2 background. Cas11−/−and wild-type mice infected withListeriahad similar hepatic microabscess formation in terms of histologic appearance, size, and number. Apoptosis ofL. monocytogenes-infected hepatocytes in vivo and in vitro in primary culture was not altered by the absence of Cas11. Serum IL-18 and IL-1β levels were similar in Cas11−/−mice and controls. Endotoxin (lipopolysaccharide [LPS])-challenged Cas11−/−mice were deficient in the production of gamma interferon. IL-1β responses in Cas11−/−were normal with intravenous administration of LPS but decreased with intraperitoneal administration. Our findings suggest that Cas11 deficiency does not impair the immune response to infection withL. monocytogenes. Apoptosis and maturation of IL-18 and IL-1β were normal despite Cas11 deficiency. LPS-induced proinflammatory pathways are altered by the absence of Cas11. While Cas11-mediated Cas1 and Cas3 activation is crucial for cytokine maturation and apoptosis during inflammation, alternative pathways allow normal inflammatory and apoptotic responses during infection withL. monocytogenes.
Inhibition of Fusion ofChlamydia trachomatisInclusions at 32°C Correlates with Restricted Export of IncA
Authors: Fields K. A. , Fischer E. , Hackstadt T.
Published: July 2002

Chlamydia trachomatisis an obligate intracellular bacterium that develops within a parasitophorous vacuole termed an inclusion. The inclusion is nonfusogenic with lysosomes but intercepts lipids from a host cell exocytic pathway. Initiation of chlamydial development is concurrent with modification of the inclusion membrane by a set ofC. trachomatis-encoded proteins collectively designated Incs. One of these Incs, IncA, is functionally associated with the homotypic fusion of inclusions. Inclusions also do not fuse when cultures are multiply infected withC. trachomatisand cultivated at 32°C. We obtained evidence linking these experimental observations by characterizing IncA localization in 32°C cultures. Analysis of inclusions by light and transmission electron microscopy confirmed that HeLa cells infected with multipleC. trachomatiselementary bodies and cultivated at 32°C for 24 h contained multiple, independent inclusions. Reverse transcriptase PCR and immunoblot analyses ofC. trachomatis-infected HeLa cells demonstrated the presence of IncA at 24 h in 32°C cultures. When parallel cultures were probed with IncA-specific antibodies in indirect immunofluorescence assays, IncA was detectable in intracellular chlamydiae but not within the inclusion membrane. In addition, analysis of purified reticulate bodies from 37 and 32°C cultures showed that bacterium-associated pools of IncA are enriched in cultures grown at 32°C. Microscopic observation of infected cells revealed that some vacuoles had fused by 48 h postinfection, and this finding was correlated with the detection of IncA in inclusion membranes by immunofluorescence microscopy. The data are consistent with a requirement for IncA in fusions ofC. trachomatisinclusions and suggest that the effect of incubation at 32°C is manifested by restricted export of IncA to the inclusion membrane.
LuxR- and Acyl-Homoserine-Lactone-Controlled Non-
lux
Genes Define a Quorum-Sensing Regulon in
Vibrio fischeri
Authors: Callahan Sean M. , Dunlap Paul V.
Published: July 2002

The luminescence (
lux
) operon (
luxICDABEG
) of the symbiotic bacterium
Vibrio fischeri
is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the
luxI
-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the
ainS
-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by
lux
genes, we examined the protein patterns of
V. fischeri
quorum-sensing mutants defective in
luxI
,
ainS
, and
luxR
by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized:
qsrP
directs cells to synthesize an apparently novel periplasmic protein, and
ribB
is a homolog of the
Escherichia coli
gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The
qsrP
and
ribB
promoter regions each contained a sequence similar to the
lux
operon
lux
box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation of
lux
operon transcription.
V. fischeri qsrP
and
ribB
mutants exhibited no distinct phenotype in culture. However, a
qsrP
mutant, in competition with its parent strain, was less successful in colonizing
Euprymna scolopes
, the symbiotic host of
V. fischeri
. The newly identified QSR genes, together with the
lux
operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in
V. fischeri.
A Mutation in
secY
That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation
Authors: Matsumoto Gen , Homma Takayuki , Mori Hiroyuki , Ito Koreaki
Published: July 2002

A cold-sensitive
secY
mutant (
secY125
) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export. Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein. The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions. These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration. In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired. We conclude that this periplasmic
secY
mutation causes unbalanced early and late functions in translocation, compromising the translocase's ability to catalyze multiple rounds of reactions.
Glucose Transporter Mutants of
Escherichia coli
K-12 with Changes in Substrate Recognition of IICB
Glc
and Induction Behavior of the
ptsG
Gene
Authors: Zeppenfeld Tim , Larisch Christina , Lengeler Joseph W. , Jahreis Knut
Published: July 2002

In
Escherichia coli
K-12, the major glucose transporter with a central role in carbon catabolite repression and in inducer exclusion is the phosphoenolpyruvate-dependent glucose:phosphotransferase system (PTS). Its membrane-bound subunit, IICB
Glc
, is encoded by the gene
ptsG
; its soluble domain, IIA
Glc
, is encoded by
crr
, which is a member of the
pts
operon. The system is inducible by
d
-glucose and, to a lesser degree, by
l
-sorbose. The regulation of
ptsG
transcription was analyzed by testing the induction of IICB
Glc
transporter activity and of a single-copy Φ(
ptsGop-lacZ
) fusion. Among mutations found to affect directly
ptsG
expression were those altering the activity of adenylate cyclase (
cyaA
), the repressor DgsA (
dgsA
; also called Mlc), the general PTS proteins enzyme I (
ptsI
) and histidine carrier protein HPr (
ptsH
), and the IIA
Glc
and IIB
Glc
domains, as well as several authentic and newly isolated UmgC mutations. The latter, originally thought to map in the repressor gene
umgC
outside the
ptsG
locus, were found to represent
ptsG
alleles. These affected invariably the substrate specificity of the IICB
Glc
domain, thus allowing efficient transport and phosphorylation of substrates normally transported very poorly or not at all by this PTS. Simultaneously, all of these substrates became inducers for
ptsG
. From the analysis of the mutants, from
cis-trans
dominance tests, and from the identification of the amino acid residues mutated in the UmgC mutants, a new regulatory mechanism involved in
ptsG
induction is postulated. According to this model, the phosphorylation state of IIB
Glc
modulates IIC
Glc
which, directly or indirectly, controls the repressor DgsA and hence
ptsG
expression. By the same mechanism, glucose uptake and phosphorylation also control the expression of the
pts
operon and probably of all operons controlled by the repressor DgsA.
A Large Gene Cluster for the
Clostridium cellulovorans
Cellulosome
Authors: Tamaru Yutaka , Karita Shuichi , Ibrahim Atef , Chan Helen , Doi Roy H.
Published: July 2002

A large gene cluster for the
Clostridium cellulovorans
cellulosome has been cloned and sequenced upstream and downstream of the
cbpA
and
exgS
genes (C.-C. Liu and R. H. Doi, Gene 211:39–47, 1998). Gene walking revealed that the
engL
gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244–247, 2000) was located downstream of the
cbpA-exgS
genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is
cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN
and is about 22 kb in length. The
engN
gene did not have a complete catalytic domain, indicating that
engN
is a truncated gene. This large gene cluster is flanked at the 5′ end by a putative noncellulosomal operon consisting of
nifV-orf1-sigX-regA
and at the 3′ end by noncellulosomal genes with homology to transposase (
trp
) and malate permease (
mle
). Since gene clusters for the cellulosome are also found in
C. cellulolyticum
and
C. josui
, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.
OmpR Regulates the Two-Component System SsrA-SsrB in
Salmonella
Pathogenicity Island 2
Authors: Lee Anthea K. , Detweiler Corrella S. , Falkow Stanley
Published: July 2002

Salmonella
pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs,
ssrAB
(
spiR
), and genes within the secretion system, including the structural gene
ssaH
, are transcribed after
Salmonella
enters host cells. We have studied the transcriptional regulation of
ssrAB
and the secretion system by using
gfp
fusions to the
ssrA
and
ssaH
promoters. We found that early transcription of
ssrA
, after entry into macrophages, is most efficient in the presence of OmpR. An
ompR
mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the
ssrA
promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not
ompR
mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of
ssrA
soon after
Salmonella
enters the macrophage.
Isolation of an Inner Membrane-Derived Subfraction That Supports In Vitro Replication of a Mini-RK2 Plasmid in
Escherichia coli
Authors: Kim Peter D. , Firshein William
Published: July 2002

Previous results have demonstrated that the inner, but not the outer, membrane fraction of
Escherichia coli
is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19–31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428–443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind
oriV
and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766–6772, 1995).
The
Yersinia pestis
YscY Protein Directly Binds YscX, a Secreted Component of the Type III Secretion Machinery
Authors: Day James B. , Plano Gregory V.
Published: July 2002

Human pathogenic yersiniae organisms export and translocate the Yop virulence proteins and V antigen upon contact with a eukaryotic cell.
Yersinia pestis
mutants defective for production of YscX or YscY were unable to export the Yops and V antigen. YscX and YscY were both present in the
Y. pestis
cell pellet fraction; however, YscX was also found in the culture supernatant. YscY showed structural and amino acid sequence similarities to the Syc family of proteins. YscY specifically recognized and bound to a region of YscX that included a predicted coiled-coil region. These data suggest that YscY may function as a chaperone for YscX in
Y. pestis.
Oxaloacetate Synthesis in the Methanarchaeon
Methanosarcina barkeri
: Pyruvate Carboxylase Genes and a Putative
Escherichia coli
-Type Bifunctional Biotin Protein Ligase Gene (
bpl/birA
) Exhibit a Unique Organization
Authors: Mukhopadhyay Biswarup , Purwantini Endang , Kreder Cynthia L. , Wolfe Ralph S.
Published: July 2002

Evidence is presented that, in
Methanosarcina barkeri
oxaloacetate synthesis, an essential and major CO
2
fixation reaction is catalyzed by an apparent α
4
β
4
-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70°C, insensitive to aspartate and glutamate, mildly inhibited by α-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg
2+
. It showed negative cooperativity towards bicarbonate at 70°C but not at 37°C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin.
pycA, pycB
, and a putative
bpl
gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of
M. barkeri
and the closely related euryarchaeon
Archaeoglobus fulgidus
appeared to be of the
Escherichia coli
-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)Pro
X
5
Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their “swinging arms.”
Localization of NAD(P)H Dehydrogenase in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Ohkawa Hiroshi , Sonoda Masatoshi , Shibata Mari , Ogawa Teruo
Published: July 2002

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of
Synechocystis
sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.
Microaerophilic Induction of the Alpha-Crystallin Chaperone Protein Homologue (
hspX
) mRNA of
Mycobacterium tuberculosis
Authors: DesJardin Lucy E. , Hayes LaDonna G. , Sohaskey Charles D. , Wayne Lawrence G. , Eisenach Kathleen D.
Published: July 2002

Among the products that are expressed when
Mycobacterium tuberculosis
undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the
hspX
mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for
hspX
mRNA, levels of 16S rRNA,
fbpB
mRNA (encoding the 85B alpha antigen), and
aroB
mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in
hspX
mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.
Genetics and Regulation of Chitobiose Utilization in
Borrelia burgdorferi
Authors: Tilly Kit , Elias Abdallah F. , Errett Jennifer , Fischer Elizabeth , Iyer Radha , Schwartz Ira , Bono James L. , Rosa Patricia
Published: July 2002

Borrelia burgdorferi
spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of
N
-acetylglucosamine (GlcNAc). The
B. burgdorferi celA
,
celB
, and
celC
genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the
celB
gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes
chbA
,
chbB
, and
chbC
, since they probably encode a chitobiose transporter. We also found that the
chbC
gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.
Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) of
Porphyromonas gingivalis
Authors: Sroka Aneta , Sztukowska Maryta , Potempa Jan , Travis James , Genco Caroline Attardo
Published: July 2002

Porphyromonas gingivalis
can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of
P. gingivalis
(gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the β-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of
P. gingivalis
strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide
P. gingivalis
with a useable source of heme for growth and may contribute to the proliferation of
P. gingivalis
within periodontal pockets in which erythrocytes are abundant.
Characterization of Xylanolytic Enzymes in
Clostridium cellulovorans
: Expression of Xylanase Activity Dependent on Growth Substrates
Authors: Kosugi Akihiko , Murashima Koichiro , Doi Roy H.
Published: July 2002

Xylanase activity of
Clostridium cellulovorans
, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High α-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that
C. cellulovorans
is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.
Interplay between the Specific Chaperone-Like Proteins HybG and HypC in Maturation of Hydrogenases 1, 2, and 3 from
Escherichia coli
Authors: Blokesch Melanie , Magalon Axel , Böck August
Published: July 2002

The
hybG
gene product from
Escherichia coli
has been identified as a chaperone-like protein acting in the maturation of hydrogenases 1 and 2. It was shown that HybG forms a complex with the precursor of the large subunit of hydrogenase 2. As with HypC, which is the chaperone-like protein involved in hydrogenase 3 maturation, the N-terminal cysteine residue is crucial for complex formation. Introduction of a deletion into
hybG
abolished the generation of active hydrogenase 2 but only quantitatively reduced hydrogenase 1 activity since HypC could replace HybG in this function. In contrast, HybG could not take over the role of HypC in a Δ
hypC
genetic background. Overproduction of HybG, especially of the variants with the replaced N-terminal cysteine residue, strongly interfered with hydrogenase 3 maturation, apparently by titrating some other component(s) of the maturation machinery. The results indicate that the three hydrogenase isoenzymes not only are interacting at the functional level but are also interconnected during the maturation process.
Metabolic Flux Responses to Pyruvate Kinase Knockout in
Escherichia coli
Authors: Emmerling Marcel , Dauner Michael , Ponti Aaron , Fiaux Jocelyne , Hochuli Michel , Szyperski Thomas , Wüthrich Kurt , Bailey J. E. , Sauer Uwe
Published: July 2002

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient
Escherichia coli
was estimated using biosynthetically directed fractional
13
C labeling experiments with [U-
13
C
6
]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in
E. coli
was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient
E. coli
, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in
E. coli
is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.
Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of
Escherichia coli
Authors: Jackson Debra W. , Suzuki Kazushi , Oakford Lawrence , Simecka Jerry W. , Hart Mark E. , Romeo Tony
Published: July 2002

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of
Escherichia coli
K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the
E. coli
K-12
csrA
gene repressed biofilm formation by related bacterial pathogens. A
csrA
knockout mutation enhanced biofilm formation in
E. coli
strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this
csrA
mutation did not affect biofilm formation by a
glgA
(glycogen synthase) knockout mutant. Complementation studies with
glg
genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded
csrA
′
-
′
lacZ
translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of
E. coli
biofilm development.
The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in
Escherichia coli
Authors: Conter Annie , Sturny Rachel , Gutierrez Claude , Cam Kaymeuang
Published: July 2002

The RcsCB His-Asp phosphorelay system regulates the expression of several genes of
Escherichia coli
, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of
E. coli
with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in
rcsB
or
rcsC
mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.
Roles of Internal Cysteines in the Function, Localization, and Reactivity of the TraV Outer Membrane Lipoprotein Encoded by the F Plasmid
Authors: Harris Robin L. , Silverman Philip M.
Published: July 2002

We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutanttraVgenes:traVC10S,traVC18S, andtraVC10S/C18S. All three cysteine mutations complemented atraVmutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of thetraVC18Sand, especially,traVC10S/C18Smutant strains were significantly less than those of thetraV+andtraVC10Sstrains. Thus, unlike theAgrobacterium tumefaciensT-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraVC18Smutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraVC10Sor TraVC10S/C18Sproteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraVC10Sand TraVC18Sproteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraVC10S/C18Sprotein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.
Molecular and Conventional Epidemiology of
Mycobacterium tuberculosis
in Botswana: a Population-Based Prospective Study of 301 Pulmonary Tuberculosis Patients
Authors: Lockman Shahin , Sheppard Jeffery D. , Braden Christopher R. , Mwasekaga Michael J. , Woodley Charles L. , Kenyon Thomas A. , Binkin Nancy J. , Steinman Michael , Montsho Faustina , Kesupile-Reed Matlhatso , Hirschfeldt Colette , Notha Malebogo , Moeti Themba , Tappero Jordan W.
Published: July 2002

Little is known about patterns of tuberculosis (TB) transmission among populations in developing countries with high rates of TB and human immunodeficiency virus (HIV) infection. To examine patterns of TB transmission in such a setting, we performed a population-based DNA fingerprinting study among TB patients in Botswana. Between January 1997 and July 1998, TB patients from four communities in Botswana were interviewed and offered HIV testing. Their
Mycobacterium tuberculosis
isolates underwent DNA fingerprinting using IS
6110
restriction fragment length polymorphism, and those with matching fingerprints were reinterviewed. DNA fingerprints with >5 bands were considered clustered if they were either identical or differed by at most one band, while DNA fingerprints with ≤5 bands were considered clustered only if they were identical. TB isolates of 125 (42%) of the 301 patients with completed interviews and DNA fingerprints fell into 20 different clusters of 2 to 16 patients. HIV status was not associated with clustering. Prior imprisonment was the only statistically significant risk factor for clustering (risk ratio, 1.5; 95% confidence interval, 1.1 to 2.0). In three communities where the majority of eligible patients were enrolled, 26 (11%) of 243 patients overall and 26 (25%) of 104 clustered patients shared both a DNA fingerprint and strong antecedent epidemiologic link. Most of the increasing TB burden in Botswana may be attributable to reactivation of latent infection, but steps should be taken to control ongoing transmission in congregate settings. DNA fingerprinting helps determine loci of TB transmission in the community.
Existence of Hepatitis C Virus in
Culex quinquefasciatus
after Ingestion of Infected Blood: Experimental Approach to Evaluating Transmission by Mosquitoes
Authors: Chang Ting-Tsung , Chang Tsuey-Yu , Chen Cheng-Chen , Young Kung-Chia , Roan Jun-Neng , Lee Yen-Chien , Cheng Pin-Nan , Wu Hua-Lin
Published: July 2002

We used PCR to detect hepatitis C virus (HCV) RNA among supernatants of ground
Culex quinquefasciatus
mosquitoes that (i) had been fed HCV-positive blood, (ii) had been intrathoracically inoculated with HCV-positive blood, or (iii) were from homes of hepatitis C patients. HCV RNA was detectable under all three conditions, but it did not replicate in mosquitoes and was not detectably transmitted during feeding.
Multicenter Evaluation of Fully Automated BACTEC Mycobacteria Growth Indicator Tube 960 System for Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Bemer Pascale , Palicova Frantiska , Rüsch-Gerdes Sabine , Drugeon Henri B. , Pfyffer Gaby E.
Published: July 2002

The reliability of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system for testing of
Mycobacterium tuberculosis
susceptibility to the three front-line drugs (isoniazid [INH], rifampin [RIF], and ethambutol [EMB]) plus streptomycin (STR) was compared to that of the BACTEC 460 TB system. The proportion method was used to resolve discrepant results by an independent arbiter. One hundred and ten strains were tested with an overall agreement of 93.5%. Discrepant results were obtained for seven strains (6.4%) with INH (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for one strain (0.9%) with RIF (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for seven strains (6.4%) with EMB (six resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB; one susceptible by BACTEC MGIT 960 and resistant by BACTEC 460 TB), and for 19 strains (17.3%) with STR (resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB). After resolution of discrepant results, the sensitivity of the BACTEC MGIT 960 system was 100% for all four drugs and specificity ranged from 89.8% for STR to 100% for RIF. Turnaround times were 4.6 to 11.7 days (median, 6.5 days) for BACTEC MGIT 960 and 4.0 to 10.0 days (median, 7.0 days) for BACTEC 460 TB. These data demonstrate that the fully automated and nonradiometric BACTEC MGIT 960 system is an accurate method for rapid susceptibility testing of
M. tuberculosis
.
Polymorphisms of
Helicobacter pylori
HP0638 Reflect Geographic Origin and Correlate with
cagA
Status
Authors: Ando T. , Peek R. M. , Pride D. , Levine S. M. , Takata T. , Lee Y.-C. , Kusugami K. , van der Ende A. , Kuipers E. J. , Kusters J. G. , Blaser M. J.
Published: July 2002

Since the associations between
Helicobacter pylori
genotype and disease differ in Asia and the West, we investigated the correlation between HP0638, encoding an outer membrane protein, and potential markers of virulence (
cagA
,
vacA
, and
iceA
). For 109 strains from nine countries, the status of
cagA
,
vacA
, and
iceA
was determined by PCR and/or a line probe assay. We also studied 18 strains from 8 patients (parents and 6 daughters) from a Dutch family and paired strains collected on average 8 years apart from 11 patients. When the HP0638 signal sequences were amplified by PCR and DNA sequence determinations were performed, 89 (96%) of 93
cagA
-positive strains had HP0638 in frame, versus none (0%) of 16
cagA
-negative strains (
P
< 0.001). Among strains in which HP0638 was in frame, a six-CT dinucleotide repeat pattern was dominant in Western countries (23 of 33 strains [70%]), while a pattern of three CT repeats with another CT after four T’s (3 + 1-CT-repeat pattern) was dominant in East Asia (31 of 46 strains [67%]); however, specific CT repeat patterns did not correlate with clinical outcome. HP0638 phylogenetic trees also showed geographic characters. The HP0638 frame status and CT dinucleotide repeat patterns were identical for 9 of 11 pairs of strains obtained on average 8 years apart from individuals and the 15 strains obtained from the mother and all six daughters. Thus, HP0638 frame status and
cagA
status are strongly correlated. The CT dinucleotide repeat pattern in the putative HP0638 signal sequence has geographic characters and appears stable in particular patients and families over a period of years. Analysis of HP0638 CT polymorphisms may serve as a new typing system to discriminate
H
.
pylori
isolates for epidemiological purposes.
Molecular Epidemiology and Drug Resistance of
Mycobacterium tuberculosis
Isolates from Ethiopian Pulmonary Tuberculosis Patients with and without Human Immunodeficiency Virus Infection
Authors: Bruchfeld Judith , Aderaye Getachew , Palme Ingela Berggren , Bjorvatn Bjarne , Ghebremichael Solomon , Hoffner Sven , Lindquist Lars
Published: July 2002

We have analyzed the molecular epidemiology and drug resistance of 121
Mycobacterium tuberculosis
isolates from consecutive patients with culture-positive pulmonary tuberculosis attending a university hospital outpatient department in Addis Ababa, Ethiopia. Restriction fragment length polymorphism analysis and spoligotyping were used to analyze the DNA fingerprinting patterns. Fifty-one (41.2%) of the isolates were found in 13 clusters with two or more identical DNA patterns. Two such clusters contained 49.0% of all clustered isolates. In a multivariate logistic regression model, human immunodeficiency virus (HIV)-positive serostatus was significantly associated with clustering of isolates for patients of both sexes (odds ratio [OR], 2.55; 95% confidence interval [CI], 1.17 to 5.80). There was a trend toward increased clustering of isolates from tuberculous women residing in Addis Ababa (OR, 2.10; 95% CI, 0.85 to 5.25). In total, 17 of 121 isolates (14.0%) were resistant to one or more of the antituberculosis drugs isoniazid (8.3%), streptomycin (7.4%), rifampin (2.5%), and ethambutol (1.7%). The high rate of drug-resistant isolates (29.6%) coincided with the peak prevalence of HIV infection (77.8%) in patients 35 to 44 years old. The majority (62.5%) of resistant isolates in this group were found within clusters. The simultaneous accumulation of certain bacterial clones in a patient population likely reflects recent transmission. Hence, we conclude that tuberculosis is commonly caused by recent infection with
M
.
tuberculosis
in HIV-positive Ethiopian patients. Furthermore, with the rapidly increasing prevalence of HIV infection in Ethiopia, the burden of tuberculosis, including drug-resistant tuberculosis, is likely to increase. Strengthening of classical tuberculosis control measures by promoting active case finding among HIV-positive adults with tuberculosis is warranted to reduce rates of transmission.
Epstein-Barr Virus gH Is Essential for Penetration of B Cells but Also Plays a Role in Attachment of Virus to Epithelial Cells
Authors: Molesworth Sara J. , Lake Cathleen M. , Borza Corina M. , Turk Susan M. , Hutt-Fletcher Lindsey M.
Published: July 2002

Entry of Epstein-Barr virus (EBV) into B cells is initiated by attachment of glycoprotein gp350 to the complement receptor type 2 (CR2). A complex of three glycoproteins, gH, gL, and gp42, is subsequently required for penetration. Gp42 binds to HLA class II, which functions as an entry mediator or coreceptor and, by analogy with other herpesviruses, gH is then thought to be involved virus-cell fusion. However, entry of virus into epithelial cells is thought to be different. It can be initiated by attachment by an unknown glycoprotein in the absence of CR2. There is no interaction between gp42 and HLA class II and instead a distinct complex of only the two glycoproteins gH and gL interacts with a novel entry mediator. Again, by analogy with other viruses gH is thought to be critical to fusion. To investigate further the different roles of gH in infection of the two cell types and to examine its influence on the assembly of the gH-gL-gp42 complex, we constructed two viruses, one in which the gH open reading frame was interrupted by a cassette expressing a neomycin resistance gene and the gene for green fluorescent protein and one as a control in which the neighboring nonessential thymidine kinase gene was interrupted with the same cassette. Virus lacking gH exited from cells normally, although loss of gH resulted in rapid turnover of gL and gp42 as well. The virus bound normally to B lymphocytes but could not infect them unless cells and bound virus were treated with polyethylene glycol to induce fusion. In contrast, virus that lacked the gH complex was impaired in attachment to epithelial cells and the effects of monoclonal antibodies to gH implied that this resulted from loss of gH rather than other members of the complex. These results suggest a role for gH in both attachment and penetration into epithelial cells.