bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTAutoantibodies 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTSalmonellapathogenicity 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.