bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Immunocapture PCR Assay for Detection of
Campylobacter jejuni
in Foods
Authors: Waller David F. , Ogata Steven A.
Published: July 2002

The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of
Campylobacter jejuni
in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.
Anaerobic Mineralization of Toluene by Enriched Sediments with Quinones and Humus as Terminal Electron Acceptors
Authors: Cervantes Francisco J. , Dijksma Wouter , Duong-Dac Tuan , Ivanova Anna , Lettinga Gatze , Field Jim A.
Published: July 2002

The anaerobic microbial oxidation of toluene to CO
2
coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [
13
C]toluene were recovered as
13
CO
2
in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [
13
C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.
Ecology of
Thioploca
spp.: Nitrate and Sulfur Storage in Relation to Chemical Microgradients and Influence of
Thioploca
spp. on the Sedimentary Nitrogen Cycle
Authors: Zopfi Jakob , Kjær Thomas , Nielsen Lars P. , Jørgensen Bo Barker
Published: July 2002

Microsensors, including a recently developed NO
3
−
biosensor, were applied to measure O
2
and NO
3
−
profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of
Thioploca
spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O
2
, high NO
3
−
, and bottom water current. On addition of NO
3
−
and NO
2
−
,
Thioploca
spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with
Thioploca
, the penetration depth of NO
3
−
was only 0.5 mm and a sharp maximum of NO
3
−
uptake was observed 0.5 mm above the sediment surface. In sediments with only few
Thioploca
spp., NO
3
−
was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N
2
O) was observed, which is consistent with the observation that
Thioploca
does not denitrify but reduces intracellular NO
3
−
to NH
4
+
. Measurements of the intracellular NO
3
−
and S
0
pools in
Thioploca
filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO
3
−
were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of
Thioploca
spp. and nitrate transport into the sediment.
Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery
Pediococcus
Isolates
Authors: Barney Michael , Volgyi Antonia , Navarro Alfonso , Ryder David
Published: July 2002

A total of 46 brewery and 15 ATCC
Pediococcus
isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as
Pediococcus damnosus
using
Eco
RI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18
P. damnosus
brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as
P. parvulus
, two were identified as
P. acidilactici
, and two were identified as unique
Pediococcus
species. The use of alternate restriction endonucleases indicated that
Pst
I and
Pvu
II could further differentiate some strains having identical
Eco
RI profiles. An acid-resistant
P. damnosus
isolate could be distinguished from non-acid-resistant varieties of the same species using
Pst
I instead of
Eco
RI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery
P. damnosus
isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the
P. acidilactici
reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test
Pediococcus
isolates, riboprinting proved to be a better method for subspecies differentiation.
Expression of Six Peptidases from
Lactobacillus helveticus
in
Lactococcus lactis
Authors: Luoma Susanna , Peltoniemi Kirsi , Joutsjoki Vesa , Rantanen Terhi , Tamminen Marja , Heikkinen Inka , Palva Airi
Published: July 2002

For development of novel starter strains with improved proteolytic properties, the ability of
Lactococcus lactis
to produce
Lactobacillus helveticus
aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into
L. lactis
MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the
L. helveticus
aminopeptidase genes
pepN, pepC
, and
pepX
are expressed under the control of their own promoters in
L. lactis
. The highest expression level, using a low-copy-number vector, was obtained with the
L. helveticus pepN
gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type
L. lactis
. The
L. helveticus pepI
gene, residing as a third gene in an operon in its host, was expressed in
L. lactis
under the control of the
L. helveticus pepX
promoter. The genetic background of the
L. lactis
derivatives tested did not affect the expression level of any of the
L. helveticus
peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the
L. helveticus pepD
and
pepR
clones under the control of their own promoters were below the detection limit in
L. lactis
. However, substantial amounts of recombinant pepD and PepR activities were obtained in
L. lactis
when
pepD
and
pepR
were expressed under the control of the inducible lactococcal
nisA
promoter at an optimized nisin concentration.
Mycobacterium
Diversity and Pyrene Mineralization in Petroleum-Contaminated Soils
Authors: Cheung Pui-Yi , Kinkle Brian K.
Published: July 2002

Degradative strains of fast-growing
Mycobacterium
spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using
Mycobacterium
-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous
Mycobacterium
community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer
Mycobacterium
phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil
Mycobacterium
community. Mineralization studies using [
14
C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader
Mycobacterium
sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous
Mycobacterium
populations during incubation.
Enrichment of High-Affinity CO Oxidizers in Maine Forest Soil
Authors: Hardy Kathleen R. , King Gary M.
Published: July 2002

Carboxydotrophic activity in forest soils was enriched by incubation in a flowthrough system with elevated concentrations of headspace CO (40 to 400 ppm). CO uptake increased substantially over time, while the apparent

K
m

(
app

K
m

) for uptake remained similar to that of unenriched soils (<10 to 20 ppm). Carboxydotrophic activity was transferred to and further enriched in sterile sand and forest soil. The
app

K
m

s for secondary and tertiary enrichments remained similar to values for unenriched soils. CO uptake by enriched soil and freshly collected forest soil was inhibited at headspace CO concentrations greater than about 1%. A novel isolate, COX1, obtained from the enrichments was inhibited similarly. However, in contrast to extant carboxydotrophs, COX1 consumed CO with an
app

K
m

of about 15 ppm, a value comparable to that of fresh soils. Phylogenetic analysis based on approximately 1,200 bp of its 16S rRNA gene sequence suggested that the isolate is an α-proteobacterium most closely related to the genera
Pseudaminobacter, Aminobacter
, and
Chelatobacter
(98.1 to 98.3% sequence identity).
Effect of
Bacillus thuringiensis
Cry1 Toxins in Insect Hemolymph and Their Neurotoxicity in Brain Cells of
Lymantria dispar
Authors: Cerstiaens Anja , Verleyen Peter , Van Rie Jeroen , Van Kerkhove Emmy , Schwartz Jean-Louis , Laprade Raynald , De Loof Arnold , Schoofs Liliane
Published: July 2002

Little information is available on the systemic effects of
Bacillus thuringiensis
toxins in the hemocoel of insects. In order to test whether
B. thuringiensis
-activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of
Lymantria dispar
(Lepidoptera) and
Neobellieria bullata
(Diptera) larvae. Injection of Cry1C was highly toxic to the
Lymantria
larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to
N. bullata
larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of
Lymantria
in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of
Lymantria
brain cells resulted in rapid lysis of the cultured neurons.
Identification of a New Plasmid-Encoded
sec
-Dependent Bacteriocin Produced by
Listeria innocua
743
Authors: Kalmokoff M. L. , Banerjee S. K. , Cyr T. , Hefford M. A. , Gleeson T.
Published: July 2002

Listeria innocua
743 produces an inhibitory activity demonstrating broad-spectrum inhibition of
Listeria monocytogenes
isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn
917
into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (
lisA
) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (
lisB
). Translation of the bacteriocin gene (
lisA
) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into
lisA
were sensitive to the inhibitor, indicating that
lisAB
forms a single operon and that
lisB
represents the immunity protein. Cloning of an amplicon containing the
lisAB
operon into
Escherichia coli
resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is
sec
dependent. This is the first confirmation of bacteriocin production in a
Listeria
spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.
Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of
Streptomyces coelicolor
A3(2)
Authors: Rodrı́guez E. , Banchio C. , Diacovich L. , Bibb M. J. , Gramajo H.
Published: July 2002

Two genes,
accB
and
accE
, that form part of the same operon, were cloned from
Streptomyces coelicolor
A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of
accB
and
accE
in
Escherichia coli
and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an
accB
null mutant and the thiostrepton growth dependency of a
tipAp accB
conditional mutant confirmed that AccB is essential for
S. coelicolor
viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109–3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites.
Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered
Escherichia coli
Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase
Authors: Wang Aijun A. , Mulchandani Ashok , Chen Wilfred
Published: July 2002

A genetically engineered
Escherichia coli
cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.
Plasmodium vivax
Polymorphism in a Clinical Drug Trial
Authors: Adak T. , Valecha Neena , Sharma V. P.
Published: July 2002

Data from a double-blind randomized clinical drug trial were analyzed to find the comparative responses of two antirelapse drugs, bulaquine and primaquine, against different relapsing forms of
Plasmodium vivax
infection. A 1-year follow-up study strongly suggests that the duration of preerythrocytic development of
P. vivax
is a polymorphic characteristic, exhibited by two strains of hypnozoites responsible for early and late manifestations after primary infection. Short-term relapses were significantly higher in the first half year than long-term relapses, and the reverse was true in the second half year. Clinical drug response data showed that the hypnozoites characterized for short-term relapse were not susceptible to either of the antirelapse drugs in the currently administered dose, whereas hypnozoites characterized for long incubation were significantly susceptible.
Correlation of CD4
+
T-Cell Counts Estimated by an Immunocapture Technique (Capcellia) with Viral Loads in Human Immunodeficiency Virus-Seropositive Individuals
Authors: Kannangai R. , Ramalingam S. , Jesudason M. V. , Vijayakumar T. S. , Abraham O. C. , Zachariah A. , Sridharan G.
Published: July 2002

As antiretroviral therapy becomes more affordable, valid, reliable, and inexpensive laboratory tests are also needed to monitor the progression of disease in people with human immunodeficiency virus (HIV) infection. The CD4
+
T-cell counts estimated by Capcellia, an immunocapture method, and flow cytometry were compared and were correlated with HIV type 1 (HIV-1) load. There was a significant negative correlation between the HIV-1 load and CD4
+
T-cell counts estimated by flow cytometry (
r
= −0.63,
P
= <0.001) as well as between the HIV-1 load and CD4
+
T-cell counts estimated by Capcellia (
r
= −0.61,
P
= <0.001). Capcellia is a cost-effective, user-friendly assay that correlated well with HIV-1 load determinations for individuals both with and without treatment.
High-Affinity Interaction between Gram-Negative Flagellin and a Cell Surface Polypeptide Results in Human Monocyte Activation
Authors: McDermott Patrick F. , Ciacci-Woolwine Federica , Snipes James A. , Mizel Steven B.
Published: July 2002

Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176–5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from
Salmonella enterica
serovar Enteritidis,
S. enterica
serovar Typhimurium, and
Pseudomonas aeruginosa
, is an extremely potent inducer of TNF-α production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-α production (EC
50
) was obtained with 1.5 × 10
−11
M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-α-inducing activity of the protein. Although less active than the wild-type protein, a
Salmonella
flagellin mutant composed of only the central hypervariable region retained substantial TNF-α-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-α-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.
Identification and Characterization of the
Brucella abortus
Phosphoglucomutase Gene: Role of Lipopolysaccharide in Virulence and Intracellular Multiplication
Authors: Ugalde Juan E. , Czibener Cecilia , Feldman Mario F. , Ugalde Rodolfo A.
Published: July 2002

Smooth lipopolysaccharide (LPS) of
Brucella abortus
has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly. The
B. abortus
phosphoglucomutase gene (
pgm
) was cloned, sequenced, and disrupted. The gene has a high index of identity to
Agrobacterium tumefaciens pgm
but is not part of the glycogen operon. A
B. abortus
null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen. This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.
T-Cell-Dependent Antibody Response to the Dominant Epitope of Streptococcal Polysaccharide,N-Acetyl-Glucosamine, Is Cross-Reactive with Cardiac Myosin
Authors: Malkiel Susan , Liao Li , Cunningham Madeleine W. , Diamond Betty
Published: July 2002

Autoantibodies against myosin are associated with myocarditis and rheumatic heart disease. In this study, the antigenic cross-reactivity of myosin andN-acetyl-glucosamine (GlcNAc), the dominant epitope of Group A streptococcal polysaccharide, was examined. Six antimyosin monoclonal antibodies (MAbs) derived from mice with cardiac myosin-induced myocarditis were characterized. All MAbs cross-reacted with GlcNAc, mimicking a subset of MAbs derived from rheumatic carditis patients that bind both myosin and streptococcal polysaccharide. Variable (V) region gene usage was diverse, with five of six MAb heavy-chain V regions encoded by distinct members of the J558 family and the sixth encoded by a member of the VGAM3.8 family. Light-chain V-region segments were derived from the Vk1, Vk4/5, Vk10, and Vk21 families. These antimyosin, anti-GlcNac MAbs demonstrated several T-cell-dependent features: they were predominantly immunoglobulin G, were encoded by V-region genes expressed late in development, and displayed somatic mutation. A direct correlation between the extent of somatic mutation and the affinity for myosin was observed. Affinity for GlcNAc also increased with the frequency of mutation, demonstrating that affinity maturation can occur simultaneously for both self antigen and foreign antigen. Based on these observations, we immunized mice with GlcNAc coupled to bovine serum albumin and demonstrated that a T-cell-dependent response to GlcNAc leads to antimyosin reactivity. We speculate that the pathogenic antibody response in rheumatic carditis may reflect the conversion of a T-cell-independent response to GlcNAc to a T-cell-dependent cross-reactive response to GlcNAc and myosin.
Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus
Candida albicans
Authors: Nakayama Hironobu , Mio Toshiyuki , Nagahashi Shigehisa , Kokado Michiko , Arisawa Mikio , Aoki Yuko
Published: July 2002

Conventional tools for elucidating gene function are relatively scarce in
Candida albicans
, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in
C. albicans
by the tetracycline-regulatable (TR) promoters. When the sea pansy
Renilla reniformis
luciferase gene (
RLUC1
) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the
RLUC1
gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of
N
-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.
Serum Resistance in
Haemophilus ducreyi
Requires Outer Membrane Protein DsrA
Authors: Elkins Christopher , Morrow K. John , Olsen Bonnie
Published: July 2002

Haemophilus ducreyi
is resistant to killing by normal serum antibody and complement. We discovered an
H. ducreyi
outer membrane protein required for expression of serum resistance and termed it DsrA (for “ducreyi serum resistance A”). The
dsrA
locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express
dsrA
. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of
H. ducreyi
tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring
dsrA
-nonexpressing strains were complemented in
trans
with a plasmid expressing
dsrA
. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in
H. ducreyi
does not require expression of full-length LOS but does require expression of
dsrA
. The
dsrA
locus from eight additional
H. ducreyi
strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.
SspA Is Required for LethalSalmonella entericaSerovar Typhimurium Infections in Calves but Is Not Essential for Diarrhea
Authors: Tsolis Renée M. , Adams L. Garry , Hantman Michael J. , Scherer Christina A. , Kimbrough Tyler , Kingsley Robert A. , Ficht Thomas A. , Miller Samuel I. , Bäumler Andreas J.
Published: July 2002

Salmonellapathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether theSalmonella entericaserovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with asptPmutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with ansspAmutant developed diarrhea, which resolved within 10 days but did not result in mortality. ThesspAmutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.
Treponema pallidumsubsp.pertenueDisplays Pathogenic Properties Different from Those ofT. pallidumsubsp.pallidum
Authors: Wicher Konrad , Wicher Victoria , Abbruscato Frank , Baughn Robert E.
Published: July 2002

The present study described the susceptibility of C4D guinea pigs to cutaneous infection withTreponema pallidumsubsp.pertenueHaiti B strain. The general manifestations of the disease in adults and neonates differ, to a certain degree, from those induced byT. pallidumsubsp.pallidumNichols strain. Noticeable differences between the infections were reflected in the character of the skin lesions, their onset and persistence, and the kinetics of the humoral response. The incidence and dissemination of cutaneous yaws lesions in very young guinea pigs were remarkably different from the low frequency observed in a similar age group of syphilis infection, 100 versus 17%, respectively. Moreover, as opposed toT. pallidumsubsp.pallidum,T. pallidumsubsp.pertenuedoes not cross the placenta. Offspring born to yaws-infected mothers did not produce immunoglobulin M antibodies and their organs, examined by PCR and rabbit infectivity test (RIT), were all negative. Examination of a large number of tissues and organs in adult, neonate, and maternal yaws by PCR and RIT clearly demonstrated that, unlike syphilis, there was a low incidence and short persistence of the yaws pathogen in internal organs. These findings stress the dermotropic rather than the organotropic character of yaws and provide further evidence of distinctive biological and pathological differences between yaws and venereal syphilis.
Pilus-Mediated Adherence ofHaemophilus influenzaeto Human Respiratory Mucins
Authors: Kubiet Martin , Ramphal Reuben , Weber Allan , Smith Arnold
Published: July 2002

Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeableH. influenzaebind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeableH. influenzaeto human respiratory mucins. We used isogenicH. influenzaestrains with a mutation in the structural gene for pilin (hifA), a laboratoryH. influenzaestrain transformed with a type b pilus gene cluster (from strain C54), antibodies raised againstH. influenzaeHifA, andEscherichia colistrains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliatedH. influenzaeto mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence.E. colistrains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding ofH. influenzaeto human respiratory mucins.
Interaction of
Shigella flexneri
IpaC with Model Membranes Correlates with Effects on Cultured Cells
Authors: Tran Ngoc , Serfis Alexa Barnoski , Osiecki John C. , Picking Wendy L. , Coye Lisette , Davis Rebecca , Picking William D.
Published: July 2002

Invasion of enterocytes by
Shigella flexneri
requires the properly timed release of IpaB and IpaC at the host-pathogen interface; however, only IpaC has been found to possess quantifiable activities in vitro. We demonstrate here that when added to cultured cells, purified IpaC elicits cytoskeletal changes similar to those that occur during
Shigella
invasion. This IpaC effect may correlate with its ability to interact with model membranes at physiological pH and to promote entry by an
ipaC
mutant of
S. flexneri
.
Relationship between the Tsh Autotransporter and Pathogenicity of Avian
Escherichia coli
and Localization and Analysis of the
tsh
Genetic Region
Authors: Dozois Charles M. , Dho-Moulin Maryvonne , Brée Annie , Fairbrother John M. , Desautels Clarisse , Curtiss Roy
Published: July 2002

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic
Escherichia coli
(APEC) strain χ7122. The prevalence of
tsh
was investigated in 300
E. coli
isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the
tsh
-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with χ7122 and an isogenic
tsh
mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the
tsh
gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain χ7122, near the colicin V genes in most of these strains. DNA sequences flanking the
tsh
gene of strain χ7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various
E. coli
pathotypes and other pathogenic members of the
Enterobacteriaceae
. These results demonstrate that the
tsh
gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of
E. coli
for chickens in the early stages of infection.
Cryptococcus neoformansIs a Facultative Intracellular Pathogen in Murine Pulmonary Infection
Authors: Feldmesser Marta , Kress Yvonne , Novikoff Phyllis , Casadevall Arturo
Published: July 2002

To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungusCryptococcus neoformansis typically chronic. To understand the mechanism by whichC. neoformanssurvives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show thatC. neoformansis a facultative intracellular pathogen in vivo. Furthermore, our observations suggest thatC. neoformansoccupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.
Adherence of Isogenic Flagellum-Negative Mutants of
Helicobacter pylori
and
Helicobacter mustelae
to Human and Ferret Gastric Epithelial Cells
Authors: Clyne Marguerite , Ocroinin Tadhg , Suerbaum Sebastian , Josenhans Christine , Drumm Brendan
Published: July 2002

Isogenic flagellum-negative mutants of
Helicobacter pylori
and
Helicobacter mustelae
were screened for their ability to adhere to primary human and ferret gastric epithelial cells, respectively. We also evaluated the adherence of an
H. pylori
strain with a mutation in the
flbA
gene, a homologue of the
flbF/lcrD
family of genes known to be involved in the regulation of
H. pylori
flagellar biosynthesis.
H. pylori
and
H. mustelae
mutants deficient in production of FlaA or FlaB and mutants deficient in the production of both FlaA and FlaB showed no reduction in adherence to primary human or ferret gastric epithelial cells compared with the wild-type parental strains. However, adherence of the
H. pylori flbA
mutant to human gastric cells was significantly reduced compared to the adherence of the wild-type strain. These results show that flagella do not play a direct role in promoting adherence of
H. pylori
or
H. mustelae
to gastric epithelial cells. However, genes involved in the regulation of
H. pylori
flagellar biosynthesis may also regulate the production of an adhesin.
Mutational Analysis of the
Helicobacter pylori
Vacuolating Toxin Amino Terminus: Identification of Amino Acids Essential for Cellular Vacuolation
Authors: Ye Dan , Blanke Steven R.
Published: July 2002

The functional importance of the amino terminus of the
Helicobacter pylori
vacuolating cytotoxin (VacA) was investigated by analyzing the relative levels of vacuolation of HeLa cells transfected with plasmids encoding wild-type and mutant forms of the toxin. Notably, VacA's intracellular activity was found to be sensitive to small truncations and internal deletions at the toxin's amino terminus. Moreover, alanine-scanning mutagenesis revealed the first VacA point mutations (at proline 9 or glycine 14) that completely abolish the toxin's intracellular activity.
Phagocytic Cell Killing Mediated by Secreted Cytotoxic Factors ofVibrio cholerae
Authors: Punj Vasu , Zaborina Olga , Dhiman Neelam , Falzari Kim , Bagdasarian M. , Chakrabarty A. M.
Published: July 2002

Vibrio choleraestrain VB1 secretes a number of enzymes into the outside medium that utilize ATP as a substrate. Such enzymes are found in the outside medium during the mid-log phase of growth, when the optical density at 650 nm is about 0.4, and they demonstrate nucleoside diphosphate kinase (Ndk), 5′ nucleotidase, and adenylate kinase (Ak) activities. We report that the filtered growth medium ofV. cholerae, as well as the flowthrough fraction of a green Sepharose column during fractionation of the growth medium, had very little cytotoxicity by itself towards macrophages and mast cells but exhibited significant cytotoxicity in the presence of exogenous ATP. Such fractions, harboring 5′ nucleotidase, Ndk, and presumably other ATP-utilizing enzymes, demonstrated enhanced macrophage and mast cell death; periodate-oxidized-ATP (oATP)-treated macrophage and mast cells or such cells exposed to 0.1 mM Mg2+, where surface-associated P2Z receptors could not be activated, were not susceptible to subsequent ATP addition. Microscopic visualization of mast cells clearly demonstrated cell morphological changes such as swelling, vacuolization, and nuclear fragmentation following treatment with ATP and the growth medium ofV. cholerae; however, these effects were suppressed if the mast cells were pretreated with oATP. These results strongly imply that the secreted ATP-utilizing enzymes ofV. choleraemodulate the external ATP levels of the macrophage and mast cells, leading to their accelerated death, presumably through activation of P2Z receptors. Thus, development of inhibitors for such enzymes may reduce the level ofV. choleraeinfection; alternatively, mutations in such genes may eliminateV. choleraesurvival in the gut and contribute to a safer live vaccine.
Legionella pneumophila
Major Acid Phosphatase and Its Role in Intracellular Infection
Authors: Aragon Virginia , Kurtz Sherry , Cianciotto Nicholas P.
Published: July 2002

Legionella pneumophila
is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (
pilD
) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by
L. pneumophila
and that the secreted
pilD
-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/lipase, a phospholipase A, and a
p
-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn
10
-mutagenized
L. pneumophila
. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other
pilD
-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartrate-sensitive acid phosphatase. The
map
mutants grew within macrophage-like U937 cells and
Hartmannella
amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for
L. pneumophila
intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second
pilD
-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.
Evaluation of a Tetracycline-Inducible Promoter in
Staphylococcus aureus
In Vitro and In Vivo and Its Application in Demonstrating the Role of
sigB
in Microcolony Formation
Authors: Bateman B. T. , Donegan N. P. , Jarry T. M. , Palma M. , Cheung A. L.
Published: July 2002

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-
xylR
-inducible promoter, the pSpac-
lacI
system, and the arabinose-inducible P
BAD
promoter, but each of these systems has limitations in its application to
Staphylococcus aureus
. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in
S. aureus
in vitro and inside epithelial cells as well as in an animal model of infection. Using the
xyl/tetO
promoter::
gfp
uvr
fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the
sigB
gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.
Identification and Characterization of a Second Extracellular Collagen-Like Protein Made by Group A
Streptococcus
: Control of Production at the Level of Translation
Authors: Lukomski Slawomir , Nakashima Kazumitsu , Abdi Iman , Cipriano Vincent J. , Shelvin Bobby J. , Graviss Edward A. , Musser James M.
Published: July 2002

A recent study found that group A
Streptococcus
(GAS) expresses a cell surface protein with similarity to human collagen (S. Lukomski, K. Nakashima, I. Abdi, V. J. Cipriano, R. M. Ireland, S. R. Reid, G. G. Adams, and J. M. Musser, Infect. Immun. 68:6542–6553, 2000). This streptococcal collagen-like protein (Scl) contains a long region of Gly-X-X motifs and was produced by serotype M1 GAS strains. In the present study, a second member of the
scl
gene family was identified and designated
scl2
. The Scl2 protein also has a collagen-like region, which in M1 strains is composed of 38 contiguous Gly-X-X triplet motifs. The
scl2
gene was present in all 50 genetically diverse GAS strains studied. The Scl2 protein is highly polymorphic, and the number of Gly-X-X motifs in the 50 strains studied ranged from 31 in one serotype M1 strain to 79 in serotype M28 and M77 isolates. The
scl1
and
scl2
genes were simultaneously transcribed in the exponential phase, and the Scl proteins were also produced. Scl1 and Scl2 were identified in a cell-associated form and free in culture supernatants. Production of Scl1 is regulated by Mga, a positive transcriptional regulator that controls expression of several GAS virulence factors. In contrast, production of Scl2 is controlled at the level of translation by variation in the number of short-sequence pentanucleotide repeats (CAAAA) located immediately downstream of the GTG (Val) start codon. Control of protein production by this molecular mechanism has not been identified previously in GAS. Together, the data indicate that GAS simultaneously produces two extracellular human collagen-like proteins in a regulated fashion.