bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement of Autolytic Activity for Bacteriocin-Induced Lysis
Authors: Martínez-Cuesta M. Carmen , Kok Jan , Herranz Elisabet , Peláez Carmen , Requena Teresa , Buist Girbe
Published: July 2002

The bacteriocin produced by
Lactococcus lactis
IFPL105 is bactericidal against several
Lactococcus
and
Lactobacillus
strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co
2+
and sodium dodecyl sulfate). When
L. lactis
MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363
acmAΔ1
) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of
L. lactis
MG1363, or of
L. lactis
MG1363
acmAΔ1
to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of
L. lactis
MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of
L. lactis
after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.
Distribution and Diversity of Archaea Corresponding to the Limnological Cycle of a Hypersaline Stratified Lake (Solar Lake, Sinai, Egypt)
Authors: Cytryn Eddie , Minz Dror , Oremland Ronald S. , Cohen Yehuda
Published: July 2002

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the
Halobacteriaceae
family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera
Methanobacterium
and
Methanococcus
. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus
Thermoplasma
. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.
Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments
Authors: Ravenschlag Katrin , Sahm Kerstin , Knoblauch Christian , Jørgensen Bo B. , Amann Rudolf
Published: July 2002

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the
Desulfosarcina-Desulfococcus
group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the
Desulfosarcina-Desulfococcus
group. A group of clone sequences (group SVAL1) most closely related to
Desulfosarcina variabilis
(91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of
Desulfosarcina
-
Desulfococcus
cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell
−1
day
−1
), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.
Amplified Fragment Length Polymorphism Analysis of
Campylobacter jejuni
Strains Isolated from Chickens and from Patients with Gastroenteritis or Guillain-Barré or Miller Fisher Syndrome
Authors: Duim Birgitta , Ang C. Wim , van Belkum Alex , Rigter Alan , van Leeuwen Nan W. J. , Endtz Hubert P. , Wagenaar Jaap A.
Published: July 2002

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between
Campylobacter jejuni
strains infecting chickens (
n
= 54) and those causing gastroenteritis in humans (
n
= 53). In addition,
C. jejuni
strains associated with the development of Guillain-Barré syndrome (GBS) (
n
= 14) and Miller Fisher syndrome (MFS) (
n
= 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All
C. jejuni
strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that
C. jejuni
from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.
In Situ Reproductive Rate of Freshwater
Caulobacter
spp
Authors: Poindexter Jeanne S. , Pujara Kanan P. , Staley James T.
Published: July 2002

Electron microscope grids were submerged in Lake Washington, Seattle, Wash., in June 1996 as bait to which
Caulobacter
sp. swarmers would attach and on which they would then reproduce in situ. Enumeration of bands in the stalks of attached cells implied that the caulobacters were completing approximately three reproductive cycles per day. A succession of morphological types of caulobacters occurred, as well as an episode of bacteriovore grazing that slowed the accumulation of caulobacters and prevented the aging of the population.
Transformation of Triclosan by
Trametes versicolor
and
Pycnoporus cinnabarinus
Authors: Hundt Kai , Martin Dierk , Hammer Elke , Jonas Ulrike , Kindermann Markus Karl , Schauer Frieder
Published: July 2002

We investigated the ability of
Trametes versicolor
and
Pycnoporous cinnabarinus
to metabolize triclosan.
T. versicolor
produced three metabolites, 2-
O
-(2,4,4′-trichlorodiphenyl ether)-β-
d
-xylopyranoside, 2-
O
-(2,4,4′-trichlorodiphenyl ether)-β-
d
-glucopyranoside, and 2,4-dichlorophenol.
P. cinnabarinus
converted triclosan to 2,4,4′-trichloro-2′-methoxydiphenyl ether and the glucoside conjugate known from
T. versicolor
. The conjugates showed a distinctly lower cytotoxic and microbicidal activity than triclosan did.
Prevalence and Characteristics of Shiga Toxin-Producing
Escherichia coli
from Healthy Cattle in Japan
Authors: Kobayashi Hideki , Shimada Jun , Nakazawa Muneo , Morozumi Tetsuo , Pohjanvirta Tarja , Pelkonen Sinikka , Yamamoto Koshi
Published: July 2002

The prevalence of Shiga toxin-producing
Escherichia coli
(STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with
stx
-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested
stx
-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony
stx
hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic
E. coli
(EHEC)
hlyA
genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic
E. coli
adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.
Secondary Intracellular Symbiotic Bacteria in Aphids of the GenusYamatocallis(Homoptera: Aphididae: Drepanosiphinae)
Authors: Fukatsu Takema
Published: July 2002

A novel secondary intracellular symbiotic bacterium from aphids of the genusYamatocallis(subfamily Drepanosiphinae) was characterized by using molecular phylogenetic analysis, in situ hybridization, and diagnostic PCR detection. In the aphid tissues, this bacterium (tentatively designated YSMS [Yamatocallissecondary mycetocyte symbiont]) was found specifically in large cells surrounded by primary mycetocytes harboringBuchneracells. Of nine drepanosiphine aphids examined, YSMS was detected in only two species of the same genus,Yamatocallis tokyoensisandYamatocallis hirayamae. In natural populations of these aphids, YSMS was present in 100% of the individuals. Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences demonstrated that YSMS ofY. tokyoensisandY. hirayamaeconstitute a distinct and isolated clade in the γ subdivision of the classProteobacteria. No 16S rDNA sequences of secondary endosymbionts characterized so far from other aphids showed phylogenetic affinity to YSMS. Based on these results, I suggest that YSMS was acquired by an ancestor of the genusYamatocallisand has been conserved throughout the evolution of the lineage. By using the nucleotide substitution rate for 16S rDNA ofBuchneraspp., the time of acquisition of YSMS was estimated to be about 13 to 26 million years ago, in the Miocene epoch of the Tertiary period.
Pseudomonas stutzeri
Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR
Authors: Grüntzig Verónica , Nold Stephen C. , Zhou Jizhong , Tiedje James M.
Published: July 2002

We used real-time PCR to quantify the denitrifying nitrite reductase gene (
nirS
), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for
nirS
sequences that corresponded approximately to the
Pseudomonas stutzeri
species. The assay was linear from 1 to 10
6
gene copies (
r
2
= 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of
P. stutzeri nirS
abundance in environmental samples. Although
P. stutzeri
abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that
P. stutzeri
may not be a dominant marine denitrifier.
Factors Affecting Catalase Expression in
Pseudomonas aeruginosa
Biofilms and Planktonic Cells
Authors: Frederick Jesse R. , Elkins James G. , Bollinger Nikki , Hassett Daniel J. , McDermott Timothy R.
Published: July 2002

Previous work with
Pseudomonas aeruginosa
showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 μM as FeCl
3
) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.
Bovine Rotavirus Nonstructural Protein 4 Produced by
Lactococcus lactis
Is Antigenic and Immunogenic
Authors: Enouf Vincent , Langella Philippe , Commissaire Jacqueline , Cohen Jean , Corthier Gérard
Published: July 2002

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in
Escherichia coli
, but the
nsp4
gene proved to be unstable. The capacity of the generally regarded as safe organism
Lactococcus lactis
to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in
L. lactis
for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that
L. lactis
is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.
Stress and Stress-Induced Neuroendocrine Changes Increase the Susceptibility of Juvenile Oysters (
Crassostrea gigas
) to
Vibrio splendidus
Authors: Lacoste Arnaud , Jalabert Fabienne , Malham Shelagh K. , Cueff Anne , Poulet Serge A.
Published: July 2002

Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms. Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption. In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen
Vibrio splendidus
. Oysters (
Crassostrea gigas
) were challenged with a low dose of a pathogenic
V. splendidus
strain and subjected to a mechanical stress 3 days later. Both mortality and
V.
splendidus
loads increased in stressed oysters, whereas they remained low in unstressed animals. Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of
V. splendidus
in challenged oysters. These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile
C. gigas
vulnerability to
Vibrio splendidus
.
The
Sinorhizobium meliloti
Nutrient-Deprivation-Induced Tyrosine Degradation Gene
hmgA
Is Controlled by a Novel Member of the
arsR
Family of Regulatory Genes
Authors: Milcamps Anne , Struffi Paolo , de Bruijn Frans J.
Published: July 2002

The regulation of the nutrient-deprivation-induced
Sinorhizobium meliloti
homogentisate dioxygenase (
hmgA
) gene, involved in tyrosine degradation, was examined.
hmgA
expression was found to be independent of the canonical nitrogen regulation (
ntr
) system. To identify regulators of
hmgA, s
econdary mutagenesis of an
S. meliloti
strain harboring a
hmgA-luxAB
reporter gene fusion (N4) was carried out using transposon Tn
1721
. Two independent Tn
1721
insertions were found to be located in a positive regulatory gene (
nitR
), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators. NitR was found to be a regulator of
S. meliloti hmgA
expression under nitrogen deprivation conditions, suggesting the presence of a
ntr
-independent nitrogen deprivation regulatory system.
nitR
insertion mutations were shown not to affect bacterial growth, nodulation of
Medicago sativa
(alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined. Further analysis of the
nitR
locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.
Identification of Nucleotide Sequences for the Specific and Rapid Detection of
Yersinia pestis
Authors: Radnedge Lyndsay , Gamez-Chin Silvia , McCready Paula M. , Worsham Patricia L. , Andersen Gary L.
Published: July 2002

Suppression subtractive hybridization, a cost-effective approach for targeting unique DNA, was used to identify a 41.7-kb
Yersinia pestis
-specific region. One primer pair designed from this region amplified PCR products from natural isolates of
Y. pestis
and produced no false positives for near neighbors, an important criterion for unambiguous bacterial identification.
Control of Bacterial Motility by Environmental Factors in Polarly Flagellated and Peritrichous Bacteria Isolated from Lake Baikal
Authors: Soutourina O. A. , Semenova E. A. , Parfenova V. V. , Danchin A. , Bertin P.
Published: July 2002

Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA genes showed that these strains belong to two distinct genera, i.e.,
Enterobacter
and
Pseudomonas
; they were named strains 22 and Y1000, respectively. Both strains swarmed at 25°C and remained motile at low temperatures (4°C), especially the
Pseudomonas
strain, which further supports the psychrotrophic characteristics of this strain. In contrast, a strong inhibition of motility was observed at above 30°C and with a high NaCl concentration. The existence of flagellar regulatory proteins FlhDC and FleQ was demonstrated in
Enterobacter
strain 22 and
Pseudomonas
strain Y1000, respectively, and environmental conditions reduced the expression of the structural genes potentially located at the first level in the flagellar cascade in both organisms. Finally, as in
Enterobacter
strain 22, a strong reduction in the transcription of the master regulatory gene
fleQ
was observed in
Pseudomonas
strain Y1000 in the presence of novobiocin, a DNA gyrase inhibitor, suggesting a link between DNA supercoiling and motility control by environmental factors. Thus, striking similarities observed in the two organisms suggest that these processes have evolved toward a similar regulatory mechanism in polarly flagellated and laterally flagellated (peritrichous) bacteria.
A Second Quorum-Sensing System Regulates Cell Surface Properties but Not Phenazine Antibiotic Production in
Pseudomonas aureofaciens
Authors: Zhang Zhongge , Pierson Leland S.
Published: July 2002

The root-associated biological control bacterium
Pseudomonas aureofaciens
30-84 produces a range of exoproducts, including protease and phenazines. Phenazine antibiotic biosynthesis by
phzXYFABCD
is regulated in part by the PhzR-PhzI quorum-sensing system. Mutants defective in
phzR
or
phzI
produce very low levels of phenazines but wild-type levels of exoprotease. In the present study, a second genomic region of strain 30-84 was identified that, when present in
trans
, increased β-galactosidase activity in a genomic
phzB
::
lacZ
reporter and partially restored phenazine production to a
phzR
mutant. Sequence analysis identified two adjacent genes,
csaR
and
csaI
, that encode members of the LuxR-LuxI family of regulatory proteins. No putative promoter region is present upstream of the
csaI
start codon and no
lux
box-like element was found in either the
csaR
promoter or the 30-bp intergenic region between
csaR
and
csaI
. Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system. In contrast to the multicopy effects of
csaR
and
csaI
in
trans
, a genomic
csaR
mutant (30-84R2) and a
csaI
mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84. Both mutants also produced wild-type levels of protease. However, disruption of both
csaI
and
phzI
or both
csaR
and
phzR
eliminated both phenazine and protease production completely. Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation. Additionally, the CsaI
N
-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters
Chromobacterium violaceum
CV026 and
Agrobacterium tumefaciens
A136(pCF240). Inactivation of
csaR
resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components. Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2. Both phenotypes were reversed by complementation with
csaR-csaI
or by the addition of the CsaI AHL signal. Both quorum-sensing systems play a role in colonization by strain 30-84. Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a
phzR
csaR
double mutant resulted in a 47-fold decrease. These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of
P. aureofaciens
30-84.
Identification and Characterization of the Gene Cluster Involved in Chitin Degradation in a Marine Bacterium,
Alteromonas
sp. Strain O-7
Authors: Tsujibo Hiroshi , Orikoshi Hideyuki , Baba Nao , Miyahara Masahiro , Miyamoto Katsushiro , Yasuda Masahide , Inamori Yoshihiko
Published: July 2002

Alteromonas
sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the
chiA
gene. The gene cluster consisted of three different open reading frames organized in the order
chiD
,
cbp1
, and
chiA
. The
chiD
,
cbp1
, and
chiA
genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The
cbp1
and
chiD
genes were expressed in
Escherichia coli
, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when α-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)
2
from chitin. The optimum temperature and pH of ChiD were 50°C and 7.0, respectively.
Morphological Changes of Rhizobia in Peat Cultures
Authors: Feng Lu , Roughley Rodney J. , Copeland Les
Published: July 2002

Morphological changes that take place in peat cultures of several species of rhizobia were examined. These changes seemed to be associated with enhanced survival of cells in peat and after inoculation onto plastic beads, which were used as a model system for seeds. Cell wall changes, in which the periplasmic space appeared to be occluded with electron-dense material, were observed in
Rhizobium
sp. strain SU343 and
Bradyrhizobium lupini
WU425 cells after 7 and 14 days in peat, respectively. Nutrient limitation and low O
2
concentration in peat are suggested to be factors involved in the induction of the morphological changes. Polyhydroxybutyrate reserves, which were present in broth-cultured cells of both species of rhizobia, were mobilized after transfer into peat but did not appear to influence survival after inoculation onto beads. Enhanced expression of an iron-manganese superoxide dismutase was also observed after the cells were transferred into peat. We conclude that cell wall thickening in rhizobia after transfer from broth cultures into peat is an adaptive response for long-term survival under nutrient-limited conditions in peat. Cells with thickened walls may also be more resistant to other types of stress, such as that encountered on a seed surface.
Identification of a Hydrophobin Gene That is Developmentally Regulated in the Ectomycorrhizal Fungus
Tricholoma terreum
Authors: Mankel Angela , Krause Katrin , Kothe Erika
Published: July 2002

The symbiosis between ectomycorrhizal fungi and trees is an essential part of forest ecology and depends entirely on the communication between the two partners for establishing and maintaining the relationship. The identification and characterization of differentially expressed genes is a step to identifying such signals and to understanding the regulation of this process. We determined the role of hydrophobins produced by
Tricholoma terreum
in mycorrhiza formation and hyphal development. A hydrophobin was purified from culture supernatant, and the corresponding gene was identified. The gene is expressed in aerial mycelium and in mycorrhiza. By using a heterologous antiserum directed against a hydrophobin found in the aerial mycelium of
Schizophyllum commune
, we detected a hydrophobin in the symbiosis between
T. terreum
and its native pine host
Pinus sylvestris
. The hydrophobin was found in aerial mycelium of the hyphal mantle and also in the Hartig net hyphae, which form the interface between both partners. Interestingly, this was not the case in the interaction of
T. terreum
with a host of low compatibility, the spruce
Picea abies
. The differential expression with respect to host was verified at the transcriptional level by competitive PCR. The differential protein accumulation pattern with respect to host compatibility seen by immunofluorescence staining can thus be attributed at least in part to transcriptional control of the
hyd1
gene.
Inhibition of Bacterial Growth, Enterotoxin Production, and Spore Outgrowth in Strains of
Bacillus cereus
by Bacteriocin AS-48
Authors: Abriouel Hikmate , Maqueda Mercedes , Gálvez Antonio , Martínez-Bueno Manuel , Valdivia Eva
Published: July 2002

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of
Bacillus cereus
over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 μg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.
Rapid Detection of
Shigella
Species in Environmental Sewage by an Immunocapture PCR with Universal Primers
Authors: Peng Xuanxian , Luo Wen , Zhang Jianying , Wang Sanying , Lin Shengcai
Published: July 2002

In the present study, we developed a quick, highly specific method for detection of
Shigella
species by combining immunocapturing of the bacteria and a universal primer PCR. The method drastically enhances test sensitivity, and it can be used not only for identification of
Shigella
species in the environment but also for rapid detection of other pathogens.
Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition ofEscherichia coliO157:H7 ATCC 43895 by aPseudomonassp
Authors: Samelis John , Sofos John N.
Published: July 2002

Growth ofEscherichia coliO157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producingPseudomonassp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of thePseudomonasstrain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures.Pseudomonassp. inhibitedE. coliO157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition ofE. coliO157:H7 by somePseudomonasspp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.
Characterization of the Oxygen Tolerance of a Hydrogenase Linked to a Carbon Monoxide Oxidation Pathway in
Rubrivivax gelatinosus
Authors: Maness Pin-Ching , Smolinski Sharon , Dillon Anne C. , Heben Michael J. , Weaver Paul F.
Published: July 2002

A hydrogenase linked to the carbon monoxide oxidation pathway in
Rubrivivax gelatinosus
displays tolerance to O
2
. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O
2
, 79% N
2
), its H
2
evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O
2
for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O
2
inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 μM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O
2
, potential applications of this hydrogenase are discussed.
Rapid Detection of
Escherichia coli
O157:H7 with Multiplex Real-Time PCR Assays
Authors: Jothikumar Narayanan , Griffiths Mansel W.
Published: July 2002

A SYBR Green LightCycler PCR assay using a single primer pair allowed simultaneous detection of
stx1
and/or
stx2
of
Escherichia coli
O157:H7. A distinct sequence of the Shiga-like toxin genes was amplified to yield products of 227 and/or 224 bp, respectively. The two products were distinguished by melting point curve analysis.
Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses
Authors: Van Doornum G. J. J. , Slomka M. J. , Buimer M. , Groen J. , Van den Hoek J. A. R. , Cairo I. , Vyse A. , Brown D. W. G.
Published: July 2002

Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.
Rapid Detection of
Salmonella enterica
Serovar Choleraesuis in Blood Cultures by a Dot Blot Enzyme-Linked Immunosorbent Assay
Authors: Janyapoon Kritsana , Korbsrisate Sunee , Thamapa Hatairat , Thongmin Sittichai , Kanjanahareutai Suwattana , Wongpredee Niramol , Sarasombath Suttipant
Published: July 2002

A dot blot enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to phase1-c
Salmonella
was developed for the direct detection of
Salmonella enterica
serovar Choleraesuis in blood cultures. This system was applied to the identification of serovar Choleraesuis, and the results were compared with those obtained by a conventional biochemical method. It was revealed that all 12 samples identified to be infected with serovar Choleraesuis were positive on testing by the ELISA. In contrast, 77 samples infected with bacteria commonly isolated from the blood were not reactive by the ELISA. The calculated sensitivity and specificity of the established assay are 100%.
Development of a Novel In Vitro Assay (ALS Assay) for Evaluation of Vaccine-Induced Antibody Secretion from Circulating Mucosal Lymphocytes
Authors: Chang H. Sunny , Sack David A.
Published: July 2002

We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A [IgA] and IgG) for up to 4 days of incubation at 37°C with 5% CO
2
in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of prevaccine samples were significantly higher than the ALS titers. We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in postvaccination blood samples.
Simplified Assay for Measuring
Toxoplasma gondii
Immunoglobulin G Avidity
Authors: Prince Harry E. , Wilson Marianna
Published: July 2002

A
Toxoplasma gondii
immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) was developed that combines the accuracy of assays based on end point titers and the relative ease of assays based on optical density values. Like published procedures, the new assay's avidity index (AI) was based on differential
T. gondii
-specific IgG reactivity in serum-treated wells washed with urea buffer versus that in wells washed with control buffer; unlike previous assays, however, the IgG reactivity was measured quantitatively using a standard curve. The assay was evaluated using 24 IgG-positive and IgM-positive sera collected within 5 months of the onset of symptoms (recent-infection group) and 25 IgG-positive and IgM-negative sera (past-infection group). All sera in the recent-infection group exhibited AI values of <0.18, whereas all sera in the past-infection group exhibited AI values of >0.27. The AI values of the recent-infection group showed significant correlation with the number of days after the onset of symptoms. A subset of 16 sera (8 recent and 8 past) was tested using a commercially available
T. gondii
IgG avidity ELISA based on end point titration; the results of the two assays showed highly significant correlation (
R
2
= 0.9125). In addition, we confirmed and extended the findings of other investigators, showing that AI values calculated using optical density values, but not AI values calculated using quantitative IgG values, varied significantly depending on the serum dilution used. This new assay should facilitate the accurate measurement of
T. gondii
IgG avidity in a reference laboratory setting.
Comparison of Serologic Assays for Detection of Antibodies against Human Herpesvirus 8
Authors: Corchero Jose L. , Mar Eng-Chun , Spira Thomas J. , Pellett Philip E. , Inoue Naoki
Published: July 2002

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.
Augmentation of Cellular Immunity and Reduction of Influenza Virus Titer in Aged Mice FedLactobacillus caseiStrain Shirota
Authors: Hori Tetsuji , Kiyoshima Junko , Shida Kan , Yasui Hisako
Published: July 2002

We investigated whether oral administration ofLactobacillus caseistrain Shirota activates the cellular immune system and ameliorates influenza virus (IFV) titer in the nasal site in upper respiratory IFV infection by using aged mice. Natural killer activity of splenocytes and lung cells of aged mice fed anL. caseistrain Shirota diet (L.caseistrain Shirota group) was significantly (P< 0.01 andP< 0.05) increased compared to those fed a control diet (control group). The increases were 1.5- and 2.5-fold, respectively. In aged mice fed an XL.caseistrain Shirota diet, potent induction of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), which play a very important role in excluding IFV, was evident in nasal lymphocytes. IFN-γ and TNF-α production increased 12- and 3.5-fold, respectively. In this model of upper respiratory IFV infection, the titer of IFV in the nasal washings of aged mice fed anL.caseistrain Shirota diet was significantly (P< 0.05) lower than that in aged mice fed a control diet (101.6 ± 0.6and 102.2 ± 0.5, respectively). These findings suggest that oral administration ofL.caseistrain Shirota activates not only systemic cellular immunity but also local cellular immunity and that it ameliorates IFV infection.
Indeterminate Human Immunodeficiency Virus Western Blot Profiles in Ethiopians with Discordant Screening-Assay Results
Authors: Meles Hailu , Wolday Dawit , Fontanet Arnaud , Tsegaye Aster , Tilahun Tesfaye , Aklilu Mathias , Sanders Eduard , De Wit Tobias F. Rinke
Published: July 2002

The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate WB reactivity to HIV-1 proteins may occur in individuals who do not appear to be infected with HIV. The profiles of WB reactivity among Ethiopians are hardly known. Here, we describe the profiles of indeterminate WB reactivity in Ethiopians with discordant screening assays. Between 1996 and 2000, a total of 12,124 specimens were tested for HIV-1 antibodies. Overall, 1,437 (11.9%) were positive for HIV-1 antibody. Ninety-one (≈0.8%) gave equivocal results because of discordant results among the various screening assays and indeterminate WB profiles by the American Red Cross (ARC) criteria. Most (30.4%) of these indeterminate WB results were due to p24 reactivity. However, 12 samples (13.2%) displayed reactivity to p24 and gp41 or to p24 and gp120/160 proteins (positive by Centers for Disease Control and Prevention [CDC] criteria). Only two samples (2.2%) were reactive to both
env
glycoproteins gp41 and gp120/160 (positive by the World Health Organization [WHO] criteria). Of 31 WB assays initially indeterminate by the ARC criteria and with follow-up samples, 29 (93.5%) became negative when retested subsequently while 2 (6.5%) remained indeterminate for more than a year and were thus considered negative. Using CDC and WHO criteria, 6 (19.4%) and 2 (6.5%), respectively, of these WB assays would have been considered falsely positive. In addition, 17 indeterminate samples were negative when assessed by a nucleic acid-based amplification assay for HIV-1 viremia. In general, there was 97.8% concordance between the ARC and WHO criteria and 85.7% concordance between the ARC and CDC criteria for an indeterminate WB result. The ARC criteria best met the specified objectives for diagnosis in our setting.
Use of
Taenia crassiceps
Cysticercus Antigen Preparations for Detection of Antibodies in Cerebrospinal Fluid Samples from Patients with Neurocysticercosis (
Taenia solium
)
Authors: Pardini Alessandra Xavier , Peralta Regina Helena , Vaz Adelaide José , Machado Luis dos Ramos , Peralta José Mauro
Published: July 2002

Antigen extracts obtained from the vesicular fluid of
Taenia crassiceps
cysticerci and from fractions purified by affinity chromatography with the lectin concanavalin A and the glycoprotein antigen separated by electrophoresis were used for the detection of
Taenia solium
anticysticercus antibodies. The sensitivity and specificity obtained for all antigens were 100% in enzyme-linked immunosorbent assay with good reproducibility. Using immunoblotting of the three antigens, low-molecular-mass peptides (18 and 14 kDa) were characterized only in cerebrospinal fluid samples from patients with neurocysticercosis. The results confirm that antigen fractions purified from
T. crassiceps
cisticerci are important sources of specific peptides and proved to be efficient in detecting anti-
T. solium
antibodies.
Levels of Soluble CD40 Ligand (CD154) in Serum Are Increased in Human Immunodeficiency Virus Type 1-Infected Patients and Correlate with CD4+T-Cell Counts
Authors: Sipsas Nikolaos V. , Sfikakis Petros P. , Kontos Athanasios , Kordossis Theodore
Published: July 2002

CD40 ligand (CD40L or CD154) is a costimulatory molecule expressed mainly on activated CD4+T cells. Concentrations of the soluble form of CD40L (sCD40L) in serum were determined for a cohort of 77 human immunodeficiency virus type 1 (HIV-1)-infected patients before and after initiation of highly active antiretroviral treatment (HAART) by a quantitative enzyme-linked immunosorbent assay. Circulating sCD40L levels were higher by twofold in untreated patients than in healthy controls (means ± standard deviations [SD]: 1.41 ± 1.48 versus 0.69 ± 0.59 ng/ml;P< 0.001). HIV-1-infected patients classified as CD4 T-cell category 1 had significantly higher sCD40L levels than patients classified as CD4 categories 2 and 3 (mean ± SD: 2.08 ± 1.46 ng/ml versus 1.57 ± 1.58 [category 2] and 0.94 ± 1.25 ng/ml [category 3];P= 0.046), while no correlation with clinical categories A, B, and C was found. Individual serum sCD40L levels correlated with CD4+T-cell counts (P= 0.039) but not with viral load, gamma globulin levels, or acute-inflammatory-response markers. After 8 to 12 months of HAART, a further threefold increase of serum sCD40L levels, which paralleled the increase of CD4+T-cell counts, was observed. These novel findings suggest that sCD40L measurement in HIV-1-infected patients could serve as a new surrogate marker useful in the assessment of treatment efficacy, especially in settings where well-equipped laboratories and funding required for CD4+T-cell count and viral load measurements are not available.