bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, Expression, and Characterization of the
katG
Gene, Encoding Catalase-Peroxidase, from the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium
Mycobacterium
sp. Strain PYR-1
Authors: Wang Rong-Fu , Wennerstrom David , Cao Wei-Wen , Khan Ashraf A. , Cerniglia Carl E.
Published: July 2002

A 81-kDa protein from
Mycobacterium
sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of
Mycobacterium
sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as
katG
for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of
Mycobacterium fortuitum
and clustered with catalase-peroxidase proteins from other
Mycobacterium
species in a phylogenetic tree. The
katG
gene was expressed in
Escherichia coli
to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.
Sulfate Reduction and Possible Aerobic Metabolism of the Sulfate-Reducing Bacterium
Desulfovibrio oxyclinae
in a Chemostat Coculture with
Marinobacter
sp. Strain MB under Exposure to Increasing Oxygen Concentrations
Authors: Sigalevich Pavel , Baev Mark V. , Teske Andreas , Cohen Yehuda
Published: July 2002

A chemostat coculture of the sulfate-reducing bacterium
Desulfovibrio oxyclinae
together with a facultative aerobe heterotroph tentatively identified as
Marinobacter
sp. strain MB was grown under anaerobic conditions and then exposed to a stepwise-increasing oxygen influx (0 to 20% O
2
in the incoming gas phase). The coculture consumed oxygen efficiently, and no residual oxygen was detected with an oxygen supply of up to 5%. Sulfate reduction persisted at all levels of oxygen input, even at the maximal level, when residual oxygen in the growth vessel was 87 μM. The portion of
D. oxyclinae
cells in the coculture decreased gradually from 92% under anaerobic conditions to 27% under aeration. Both absolute cell numbers and viable cell counts of the organism were the same as or even higher than those observed in the absence of oxygen input. The patterns of consumption of electron donors and acceptors suggest that aerobic incomplete oxidation of lactate to acetate is performed by
D. oxyclinae
under high oxygen input. Both organisms were isolated from the same oxic zone of a cyanobacterial mat where they have to adapt to daily shifts from oxic to anoxic conditions. This type of syntrophic association may occur in natural habitats, enabling sulfate-reducing bacteria to cope with periodic exposure to oxygen.
Enzyme Diffusion from
Trichoderma atroviride
(=
T. harzianum
P1) to
Rhizoctonia solani
Is a Prerequisite for Triggering of
Trichoderma ech42
Gene Expression before Mycoparasitic Contact
Authors: Kullnig Cornelia , Mach Robert L. , Lorito Matteo , Kubicek Christian P.
Published: July 2002

A plate confrontation experiment is commonly used to study the mechanism by which
Trichoderma
spp. antagonize and parasitize other fungi. Previous work with chitinase gene expression (
ech42
) during the precontact period of this process in which cellophane and dialysis membranes separated
Trichoderma harzianum
and its host
Rhizoctonia solani
resulted in essentially opposite results. Here, we show that cellophane membranes are permeable to proteins up to at least 90 kDa in size but that dialysis membranes are not.
ech42
was expressed during the precontact stage of the confrontation between
Trichoderma atroviride
and its host only if the cellophane was placed between the two fungi. These results are consistent with enzyme diffusion from
T. atroviride
to
R. solani
generating the trigger of
ech42
gene expression.
Use of Integrated Cell Culture-PCR To Evaluate the Effectiveness of Poliovirus Inactivation by Chlorine
Authors: Blackmer Felisa , Reynolds Kelly A. , Gerba Charles P. , Pepper Ian L.
Published: July 2002

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.
Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil
Authors: Colores Gregory M. , Macur Richard E. , Ward David M. , Inskeep William P.
Published: July 2002

We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g
−1
). Addition of the surfactant at a concentration below the CMC′ (2 mg g
−1
) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g
−1
), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g
−1
) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated
Rhodococcus
and
Nocardia
populations that were displaced by
Pseudomonas
and
Alcaligenes
populations at elevated surfactant levels. Parallel cultivation studies revealed that the
Rhodococcus
population can utilize hexadecane and that the
Pseudomonas
and
Alcaligenes
populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.
Characterization of a Novel Spirochete Associated with the Hydrothermal Vent Polychaete Annelid,
Alvinella pompejana
Authors: Campbell Barbara J. , Cary S. Craig
Published: July 2002

A highly integrated, morphologically diverse bacterial community is associated with the dorsal surface of
Alvinella pompejana
, a polychaetous annelid that inhabits active high-temperature deep-sea hydrothermal vent sites along the East Pacific Rise (EPR). Analysis of a previously prepared bacterial 16S ribosomal DNA (rDNA) library identified a spirochete most closely related to an endosymbiont of the oligochete
Olavius loisae
. This spirochete phylotype (spirochete A) comprised only 2.2% of the 16S rDNA clone library but appeared to be much more dominant when the same sample was analyzed by denaturing gradient gel electrophoresis (DGGE) and the terminal restriction fragment length polymorphism procedure (12 to 18%). PCR amplification of the community with spirochete-specific primers used in conjunction with DGGE analysis identified two spirochete phylotypes. The first spirochete was identical to spirochete A but was present in only one
A. pompejana
specimen. The second spirochete (spirochete B) was 84.5% similar to spirochete A and, more interestingly, was present in the epibiont communities of all of the
A. pompejana
specimens sampled throughout the geographic range of the worm (13°N to 32°S along the EPR). The sequence variation of the spirochete B phylotype was less than 3% for the range of
A. pompejana
specimens tested, suggesting that a single spirochete species was present in the
A. pompejana
epibiotic community. Additional analysis of the environments surrounding the worm revealed that spirochetes are a ubiquitous component of high-temperature vents and may play an important role in this unique ecosystem.
Infection of
Acanthamoeba polyphaga
with
Simkania negevensis
and
S. negevensis
Survival within Amoebal Cysts
Authors: Kahane Simona , Dvoskin Bella , Mathias Mazit , Friedman Maureen G.
Published: July 2002

Simkania negevensis
, a novel microorganism belonging to the family
Simkaniaceae
in the order
Chlamydiales
, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and
S. negevensis
has been associated with respiratory illness in humans. This study reports for the first time the ability of
S. negevensis
to survive and grow inside
Acanthamoeba polyphaga
in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of
S. negevensis
and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of
S. negevensis
in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of
S. negevensis
were observed within inclusion bodies inside
A. polyphaga
. When
S. negevensis
-infected
A. polyphaga
amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and
S. negevensis
; cysts in which
S. negevensis
cells were relegated to the space between cyst walls; and cysts containing
S. negevensis
, but apparently lacking amoebal cytoplasm.
S. negevensis
within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of
S. negevensis
needs to be investigated.
Evaluation of Two Direct Plating Methods Using Nonradioactive Probes for Enumeration of
Vibrio parahaemolyticus
in Oysters
Authors: Gooch J. A. , DePaola A. , Kaysner C. A. , Marshall D. L.
Published: July 2002

Oysters (
Crassostrea virginica
) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine
Vibrio parahaemolyticus
densities at zero time and after 5, 10, and 24 h of postharvest storage at 26°C. After 24 h of storage at 26°C, oysters were transferred to a refrigerator at 3°C and then analyzed 14 to 17 days later. The
V. parahaemolyticus
numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (
tlh
), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (
r
= 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (
r
= 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total
V. parahaemolyticus
, and they were more rapid and less labor-intensive.
Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from
Rhodococcus erythropolis
DCL14
Authors: van der Vlugt-Bergmans Cécile J. B , van der Werf Mariët J.
Published: July 2002

A monoterpene ɛ-lactone hydrolase (MLH) from
Rhodococcus erythropolis
DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4
R
)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6
R
)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4
R
)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated
mlhB
(monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.
Protein
trans
-Splicing To Produce Herbicide-Resistant Acetolactate Synthase
Authors: Sun Luo , Ghosh Inca , Paulus Henry , Xu Ming-Qun
Published: July 2002

Protein splicing in
trans
has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by
trans
-splicing has not been reported previously. In this study, we used the DnaE intein from
Synechocystis
sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by
trans
-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in
Escherichia coli
. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in
trans
and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of
E. coli
with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host
E. coli
cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.
Predictive Modeling of the Shelf Life of Fish under Nonisothermal Conditions
Authors: Koutsoumanis Konstantinos
Published: July 2002

The behavior of the natural microflora of Mediterannean gilt-head seabream (
Sparus aurata
) was monitored during aerobic storage at different isothermal conditions from 0 to 15°C. The growth data of pseudomonads, established as the specific spoilage organisms of aerobically stored gilt-head seabream, combined with data from previously published experiments, were used to model the effect of temperature on pseudomonad growth using a Belehradek type model. The nominal minimum temperature parameters of the Belehradek model (
T
min
) for the maximum specific growth rate (μ
max
) and the lag phase (
t
Lag
) were determined to be −11.8 and −12.8°C, respectively. The applicability of the model in predicting pseudomonad growth on fish at fluctuating temperatures was evaluated by comparing predictions with observed growth in experiments under dynamic conditions. Temperature scenarios designed in the laboratory and simulation of real temperature profiles observed in the fish chill chain were used. Bias and accuracy factors were used as comparison indices and ranged from 0.91 to 1.17 and from 1.11 to 1.17, respectively. The average percent difference between shelf life predicted based on pseudomonad growth and shelf life experimentally determined by sensory analysis for all temperature profiles tested was 5.8%, indicating that the model is able to predict accurately fish quality in real-world conditions.
Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR
Authors: Tajima K. , Aminov R. I. , Nagamine T. , Matsui H. , Nakamura M. , Benno Y.
Published: July 2002

A set of PCR primers was designed and validated for specific detection and quantification of
Prevotella ruminicola
,
Prevotella albensis
,
Prevotella bryantii
,
Fibrobacter succinogenes
,
Selenomonas ruminantium-Mitsuokella multiacida
,
Streptococcus bovis
,
Ruminococcus flavefaciens
,
Ruminobacter amylophilus
,
Eubacterium ruminantium
,
Treponema bryantii
,
Succinivibrio dextrinosolvens
, and
Anaerovibrio lipolytica
. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria,
F. succinogenes
and
R. flavefaciens
, were in agreement with those of earlier, culture-based experiments. The quantity of
F. succinogenes
DNA
,
predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The
R. flavefaciens
DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of
P. ruminicola
increased 7-fold and that of
P. bryantii
increased 263-fold. On day 28, the quantity of
P. ruminicola
DNA decreased 3-fold, while
P. bryantii
DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium,
E. ruminantium,
dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete,
T. bryantii,
decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in
A. lipolytica
DNA were not statistically significant. After an initial slight increase in
S. dextrinosolvens
DNA on day 3, this DNA was not detected at the end of the experiment.
S. bovis
DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of
S. ruminantium
-
M. multiacida
DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.
Identification and Functional Characterization of CbaR, a MarR-Like Modulator of the
cbaABC
-Encoded Chlorobenzoate Catabolism Pathway
Authors: Providenti Miguel A. , Wyndham R. Campbell
Published: July 2002

In
Comamonas testosteroni
BR60 (formerly
Alcaligenes
sp. strain BR60), catabolism of the pollutant 3-chlorobenzoate (3CBA) is initiated by enzymes encoded by
cbaABC
, an operon found on composite transposon Tn
5271
of plasmid pBRC60. The
cbaABC
gene product CbaABC converts 3CBA to protocatechuate (PCA) and 5-Cl-PCA, which are then metabolized by the chromosomal PCA
meta
(extradiol) ring fission pathway. In this study,
cbaA
was found to possess a ς
70
type promoter. O
2
uptake experiments with whole cells and expression studies with
cbaA
-
lacZ
constructs showed that
cbaABC
was induced by 3CBA. Benzoate, which is not a substrate of the 3CBA pathway, was a gratuitous inducer, and CbaR, a MarR family repressor coded for by a divergently transcribed gene upstream of
cbaABC
, could modulate induction mediated by benzoate. Purified CbaR bound specifically to two regions of the
cbaA
promoter (P

cbaA

); site I, a high-affinity site, is between the transcriptional start point (position +1) and the start codon of
cbaA
, while site II, a lower-affinity site, overlaps position +1. 3CBA at concentrations as low as 40 μM interfered with binding to P

cbaA

. PCA also interfered with binding, while benzoate only weakly disrupted binding. Unexpectedly, benzoate with a hydroxyl or carboxyl at position 3 improved CbaR binding. Data are also presented that suggest that an unidentified regulator is encoded on the chromosome that induces
cbaABC
in response to benzoate and 3CBA.
Characterization ofSchizosaccharomyces pombeMalate Permease by Expression inSaccharomyces cerevisiae
Authors: Camarasa Carole , Bidard Frédérique , Bony Muriel , Barre Pierre , Dequin Sylvie
Published: July 2002

InSaccharomyces cerevisiae,l-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression inS. cerevisiaeof theMAE1gene, encodingSchizosaccharomyces pombemalate permease, markedly increasedl-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes),l-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters:Vmax= 8.7 nmol/mg/min;Km= 1.6 mM) and some simple diffusion of the undissociatedl-malic acid (Kd= 0.057 min−1). As totall-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain.l-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ΔpH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related tol-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibitedl-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. InS. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediatedl-malic acid import inS. cerevisiae, and intracellular pH remained constant. This suggests that, inS. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.
Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in
Aspergillus parasiticus
Authors: Chiou Ching-Hsun , Miller Michael , Wilson David L. , Trail Frances , Linz John E.
Published: July 2002

The
nor-1
gene in the filamentous fungus
Aspergillus parasiticus
encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on
nor-1
expression, we generated plasmid pAPGUSNNB containing a
nor-1
promoter-β-glucuronidase (GUS) (encoded by
uidA
) reporter fusion with
niaD
(encodes nitrate reductase) as a selectable marker.
niaD
transformants of
A. parasiticus
strain NR-1 (
niaD
) carried pAPGUSNNB integrated predominantly at the
nor-1
or
niaD
locus. Expression of the native
nor-1
and
nor-1
::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of
nor-1
promoter function with pAPGUSNNB integrated at
nor-1
was similar to that observed for the native
nor-1
gene. In contrast,
nor-1
promoter activity in pAPGUSNNB and a second
nor-1
::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at
niaD
. Because
niaD
-dependent regulation could account for the absence of expression at
niaD
, a third chromosomal location was analyzed using pAPGUSNP, which contained
nor-1
::GUS plus
pyrG
(encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at
nor-1
and was not detectable at
pyrG
, even under growth conditions that required
pyrG
expression.
nor-1
::GUS is regulated similarly to the native
nor-1
gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native
nor-1
levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure
nor-1
promoter activity and that
nor-1
is subject to position-dependent regulation in the
A. parasiticus
chromosome.
Isogenic Strain of
Escherichia coli
O157:H7 That Has Lost both Shiga Toxin 1 and 2 Genes
Authors: Feng Peter , Dey Manashi , Abe Akio , Takeda Tae
Published: July 2002

An
Escherichia coli
O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit β-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93
gusA
mutation, the cluster I
ehxA
gene for enterohemolysin, and the
eae
gene for γ-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of
Xba
I-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both
stx
genes.
Use of Recombinant BP26 Protein in Serological Diagnosis of
Brucella melitensis
Infection in Sheep
Authors: Cloeckaert Axel , Baucheron Sylvie , Vizcaino Nieves , Zygmunt Michel S.
Published: July 2002

Previously a
Brucella
protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and
B. melitensis
Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in
Escherichia coli
in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (
n
= 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (
n
= 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (
n
= 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (
n
= 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with
B. melitensis
H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and
B. melitensis
Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a
B. melitensis
Rev.1
bp26
gene deletion mutant should be constructed to ensure this differentiation.
Toxin Gene Analysis of a Variant Strain of
Clostridium difficile
That Causes Human Clinical Disease
Authors: Sambol Susan P. , Merrigan Michelle M. , Lyerly David , Gerding Dale N. , Johnson Stuart
Published: July 2002

A toxin variant strain of
Clostridium difficile
was isolated from two patients with
C. difficile
-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for
C. difficile
toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (
tcdA
) and toxin B (
tcdB
) genes of type CF2 isolate 5340. Type CF2 5340
tcdA
exhibited a 1,821-bp truncation, due to three deletions in the 3′ end of the gene, and a point mutation in the 5′ end of the gene, resulting in a premature stop codon at
tcdA
position 139. Type CF2 5340
tcdB
exhibited multiple nucleotide base substitutions in the 5′ end of the gene compared to
tcdB
of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant
C. difficile
strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3′ end of
tcdA
demonstrated identical 1.8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of
C. difficile
that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.
Immunomodulatory Role of Endogenous Interleukin-18 in Gamma Interferon-Mediated Resolution of ReplicativeLegionella pneumophilaLung Infection
Authors: Brieland Joan K. , Jackson Craig , Hurst Steve , Loebenberg David , Muchamuel Tony , Debets Reno , Kastelein Rob , Churakova Tatyana , Abrams John , Hare Roberta , O'Garra Anne
Published: July 2002

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-γ)-mediated resolution of replicativeLegionella pneumophilalung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (106L. pneumophilaorganisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-γ mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior toL. pneumophilainfection inhibited induction of intrapulmonary IFN-γ production but did not significantly alter resolution of replicativeL. pneumophilalung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-γ and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-γ in the lung in response toL. pneumophilaand that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.
Activation of Human Peripheral Blood Mononuclear Cells by Nonpathogenic Bacteria In Vitro: Evidence of NK Cells as Primary Targets
Authors: Haller D. , Blum S. , Bode C. , Hammes W. P. , Schiffrin E. J.
Published: July 2002

The interaction of commensal bacteria with immunocompetent cells may occur in definite compartments of the mucosal immune system, as limited translocation through the epithelial barrier cannot be excluded. In this study the stimulation of human peripheral blood mononuclear cells and purified lymphocyte subsets by nonpathogenic gram-positive lactobacilli (Lactobacillus johnsoniiandLactobacillus sakei) and gram-negativeEscherichia coliwas investigated. The various bacterial strains induced a differential cytokine pattern. WhereasL. johnsoniiandL. sakeistrongly induced gamma interferon (IFN-γ) and interleukin-12 (IL-12),E. coliand lipopolysaccharide (LPS) preferentially induced IL-10 after 16 h of stimulation. Expression of activation antigens CD69 and CD25 was observed on (CD3−CD56+) natural killer (NK) cells after stimulation of total human peripheral blood mononuclear cells. All bacteria mediated the proliferation of human peripheral blood mononuclear cells, and the strongest proliferative response was observed withL. johnsonii. Purified CD4+, CD8+, and CD19+lymphocyte subsets were not activated upon bacterial stimulation but showed normal response to a mitogenic stimulus. In contrast, purified NK cells upregulated the IL-2Rα chain (CD25) and underwent proliferation when stimulated byL. johnsonii. E. coliand LPS were less effective in inducing proliferation. Expression of CD25 or secretion of IFN-γ from purified NK cells was significantly increased in the presence of bacterially primed macrophages, indicating that full activation required both bacterium- and cell contact-based signals derived from accessory cells.
Role of Group A Streptococcal Virulence Factors in Adherence to Keratinocytes
Authors: Darmstadt Gary L. , Mentele Laurel , Podbielski Andreas , Rubens Craig E.
Published: July 2002

To evaluate the role of putative group A streptococcal virulence factors in the initiation of skin infections, we compared the adherence of a wild-type M49-protein skin-associated strain to that of a series of 16 isogenic mutants created by insertional inactivation of virulence genes. None of the mutants, including the M-protein-deficient (emmmutant) strain, displayed reduced adherence to early-passage cultured human keratinocytes, but adherence of the mutant lacking hyaluronic acid capsule expression (hasmutant) was increased 13-fold. In contrast, elimination of capsule expression in M2-, M3-, and M18-proteinhasmutants increased adherence only slightly (1.3- to 2.3-fold) compared to their respective wild-type strains. A mutant with inactivation of bothemmandhasdisplayed high-level adherence (34.9 ± 4.1%) equal to that of thehasmutant strain (40.7 + 8.0%), confirming the lack of involvement of M49 protein in attachment. Moreover, adherence of the M49-protein-deficient (emmmutant) and wild-type strains was increased to the same level (57 and 55%, respectively) following enzymatic digestion of their hyaluronic acid capsule. Adherence of mutants lacking oligopeptide permease (Opp) expression was increased 3.8- to 5.5-fold, in association with decreased cell-associated hyaluronic acid capsule. Finally, soluble CD46 failed to inhibit adherence of M49- and M52-serotype skin strains. We conclude that (i) bacterial M protein and keratinocyte CD46 do not mediate adherence of M49 skin-associatedStreptococcus pyogenesto epidermal keratinocytes, (ii) hyaluronic acid capsule impedes the interaction of bacterial adhesins with keratinocyte receptors, (iii) modulation of capsule expression may be important in the pathogenesis of skin infections, and (iv) the molecular interactions in attachment of skin strains ofS. pyogenesto keratinocytes are unique and remain unidentified.
Cloning of the
Streptococcus mutans
Gene Encoding Glucan Binding Protein B and Analysis of Genetic Diversity and Protein Production in Clinical Isolates
Authors: Mattos-Graner Renata O. , Jin Song , King William F. , Chen Tsute , Smith Daniel J. , Duncan Margaret J.
Published: July 2002

Streptococcus mutans
, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The
gbpB
gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of
gbpB
genes from clinical isolates of
S. mutans
revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay.
Localization of Functional Domains of the Mitogenic Toxin of
Pasteurella multocida
Authors: Pullinger Gillian D. , Sowdhamini R. , Lax Alistair J.
Published: July 2002

The locations of the catalytic and receptor-binding domains of the
Pasteurella multocida
toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.
Identification of the
sigB
Operon in
Staphylococcus epidermidis
: Construction and Characterization of a
sigB
Deletion Mutant
Authors: Kies Stefanie , Otto Michael , Vuong Cuong , Götz Friedrich
Published: July 2002

The role of the alternative sigma factor ς
B
in
Staphylococcus epidermidis
was investigated by the construction, complementation, and characterization of a
sigB
deletion mutant. Electrophoretic analyses confirmed a profound influence of ς
B
on the expression of exoproteins and cytoplasmic proteins. Detailed investigation revealed reduced lipase and enhanced protease activity in the ς
B
mutant. Furthermore, no significant influence of ς
B
on heterologous biofilm formation or on the activity of the global regulator
agr
was detected.
Interleukin-10 StimulatesCoxiella burnetiiReplication in Human Monocytes through Tumor Necrosis Factor Down-Modulation: Role in Microbicidal Defect of Q Fever
Authors: Ghigo Eric , Capo Christian , Raoult Didier , Mege Jean-Louis
Published: July 2002

Coxiella burnetii, an obligate intracellular bacterium, is the agent of Q fever. The chronic form of the disease is associated with the overproduction of interleukin-10 and deficientC. burnetiikilling by monocytes. We hypothesized that the replication ofC. burnetiiinside monocytes requires a macrophage-deactivating cytokine such as interleukin-10. In the absence of interleukin-10,C. burnetiisurvived but did not replicate in monocytes.C. burnetiireplication (measured 15 days) was induced in interleukin-10-treated monocytes. This effect of interleukin-10 is specific since transforming growth factor β1 had no effect on bacterial replication.C. burnetiireplication involves the down-modulation of tumor necrosis factor (TNF) release. First, interleukin-10 suppressedC. burnetii-stimulated production of TNF. Second, the addition of recombinant TNF to interleukin-10-treated monocytes inhibited bacterial replication. Third, the incubation of infected monocytes with neutralizing anti-TNF antibodies favoredC. burnetiireplication. On the other hand, deficientC. burnetiikilling by monocytes from patients with chronic Q fever involves interleukin-10. Indeed,C. burnetiireplication was observed in monocytes from patients with Q fever endocarditis, but not in those from patients with acute Q fever. Bacterial replication was inhibited by neutralizing anti-interleukin-10 antibodies. As monocytes from patients with endocarditis overproduced interleukin-10, the defective bacterial killing is likely related to endogenous interleukin-10. These results suggest that interleukin-10 enables monocytes to supportC. burnetiireplication and to favor the development of chronic Q fever.
Mannan-Binding Lectin Enhances Susceptibility to Visceral Leishmaniasis
Authors: de Miranda Santos Isabel K. F. , Costa Carlos H. N. , Krieger Henrique , Feitosa Mary F. , Zurakowski David , Fardin Babak , Gomes Regis B. B. , Weiner Debra L. , Harn Donald A. , Ezekowitz R. Alan B. , Epstein Judith E.
Published: July 2002

Levels of the serum opsonin mannan-binding lectin (MBL) were directly correlated with the probability of developing visceral leishmaniasis. Monocytes infected with MBL-opsonizedLeishmania chagasipromastigotes secreted higher levels of tumor necrosis factor alpha and interleukin-6 than cells infected with nonopsonized parasites. Our findings indicate that MBL can modulate the clinical outcome of infection withL. chagasiand the function of infected macrophages.
Functional Differences among FimA Variants of
Porphyromonas gingivalis
and Their Effects on Adhesion to and Invasion of Human Epithelial Cells
Authors: Nakagawa Ichiro , Amano Atsuo , Kuboniwa Masae , Nakamura Takayuki , Kawabata Shigetada , Hamada Shigeyuki
Published: July 2002

Fimbriae of
Porphyromonas gingivalis
, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The
fimA
genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that
P. gingivalis
with type II
fimA
was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and α5β1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore,
P. gingivalis
strain HW24D1 with type II
fimA
adhered to cells and invaded them more than strains with other
fimA
genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors.
Phase Variation ofCampylobacter jejuni81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro
Authors: Guerry Patricia , Szymanski Christine M. , Prendergast Martina M. , Hickey Thomas E. , Ewing Cheryl P. , Pattarini Dawn L. , Moran Anthony P.
Published: July 2002

The outer cores of the lipooligosaccharides (LOS) of many strains ofCampylobacter jejunimimic human gangliosides in structure. A population of cells ofC. jejunistrain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2and GM3gangliosides, with minor amounts of structures mimicking GD1band GD2. Genetic analyses of genes involved in the biosynthesis of the outer core ofC. jejuni81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, anN-acetylgalactosaminyltransferase. Variation in the number of G residues withincgtAaffected the length of the open reading frame, and these changes incgtAcorresponded to a change in LOS structure from GM2to GM3ganglioside mimicry. Site-specific mutation ofcgtAin 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3ganglioside. Compared to wild-type 81-176, thecgtAmutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of theneuC1gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.
Pulmonary Inflammation Induced byPseudomonas aeruginosaLipopolysaccharide, Phospholipase C, and Exotoxin A: Role of Interferon Regulatory Factor 1
Authors: Wieland Catharina W. , Siegmund Britta , Senaldi Giorgio , Vasil Michael L. , Dinarello Charles A. , Fantuzzi Giamila
Published: July 2002

Chronic pulmonary infection withPseudomonas aeruginosais common in cystic fibrosis (CF) patients.P. aeruginosalipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, gamma interferon (IFN-γ), MIP-1α and MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-α and was a weak inducer of IL-1β, IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-γ. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-α, IL-1β, and IFN-γ than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1β and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1α and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8+and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors fromP. aeruginosainduce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.