bluuebunny/crossref_metadata_2025 huggingface dataset:
pH Regulation of Pectate Lyase Secretion Modulates the Attack ofColletotrichum gloeosporioideson Avocado Fruits
Authors: Yakoby Nir , Kobiler Ilana , Dinoor Amos , Prusky Dov
Published: July 2002

Growth ofColletotrichum gloeosporioidesin pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3.8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or whenC. gloeosporioideshyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), wherepgtranscripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americanacv. Fuerte) fruits, resistant toC. gloeosporioidesattack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant toC. gloeosporioidesattack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affectC. gloeosporioidespathogenicity. The mechanism found in avocado may have equivalents in other postharvest pathosystems and suggests new approaches for breeding against and controlling postharvest diseases.
Genetic Analysis of Chromosomal Regions of
Lactococcus lactis
Acquired by Recombinant Lytic Phages
Authors: Durmaz Evelyn , Klaenhammer Todd R.
Published: July 2002

Recombinant phages are generated when
Lactococcus lactis
subsp.
lactis
harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of
L. lactis
chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of
L. lactis
NCK203 (Abi
+
) with phage φ31.
Hin
dIII restriction maps of the variants (φ31.1, φ31.2, φ31.7, and φ31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage φ31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, φ31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and
Escherichia coli
Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the φ31.1 DNA, which replaced the φ31 origin. Three separate
Hin
dIII fragments, accounting for most of the recombinant region of φ31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.
Ionizing-Radiation Resistance in the Desiccation-Tolerant Cyanobacterium
Chroococcidiopsis
Authors: Billi Daniela , Friedmann E. Imre , Hofer Kurt G. , Caiola Maria Grilli , Ocampo-Friedmann Roseli
Published: July 2002

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10
Chroococcidiopsis
strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of
Chroococcidiopsis
strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.
Novel Scheme for Biosynthesis of Aryl Metabolites from
l
-Phenylalanine in the Fungus
Bjerkandera adusta
Authors: Lapadatescu Carmen , Giniès Christian , Le Quéré Jean-Luc , Bonnarme Pascal
Published: July 2002

Aryl metabolite biosynthesis was studied in the white rot fungus
Bjerkandera adusta
cultivated in a liquid medium supplemented with
l
-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors (
14
C- and
13
C-labelled
l
-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in
B. adusta
cultures. Metabolite formation required de novo protein biosynthesis. Our results show that
l
-phenylalanine was deaminated to
trans
-cinnamic acid by a phenylalanine ammonia lyase and
trans
-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from
l
-phenylalanine to aryl metabolites. Additionally,
trans
-cinnamic acid can also be transformed via β-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a β-oxidation degradation intermediate. To our knowledge, this is the first time that a β-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from
l
-phenylalanine is proposed.
Bacterial Symbiont Transmission in the Wood-Boring Shipworm
Bankia setacea
(Bivalvia: Teredinidae)
Authors: Sipe Alison R. , Wilbur Ami E. , Cary S. Craig
Published: July 2002

The Teredinidae (shipworms) are a morphologically diverse group of marine wood-boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid
Bankia setacea
. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the
B. setacea
gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that
B. setacea
ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement.
Comparison of Methods for Detection of
Erysipelothrix
spp. and Their Distribution in Some Australasian Seafoods
Authors: Fidalgo S. G. , Wang Q. , Riley T. V.
Published: July 2002

For many years,
Erysipelothrix rhusiopathiae
has been known to be the causative agent of the occupationally related infection erysipeloid. A survey of the distribution of
Erysipelothrix
spp. in 19 Australasian seafoods was conducted, and methodologies for the detection of
Erysipelothrix
spp. were evaluated. Twenty-one
Erysipelothrix
spp. were isolated from 52 seafood parts. Primary isolation of
Erysipelothrix
spp. was most efficiently achieved with brain heart infusion broth enrichment followed by subculture onto a selective brain heart infusion agar containing kanamycin, neomycin, and vancomycin after 48 h of incubation. Selective tryptic soy broth, with 48 h of incubation, was the best culture method for the detection of
Erysipelothrix
spp. with PCR. PCR detection was 50% more sensitive than culture.
E. rhusiopathiae
was isolated from a variety of different fish, cephalopods, and crustaceans, including a Western rock lobster (
Panulirus cygnus
). There was no significant correlation between the origin of the seafoods tested and the distribution of
E. rhusiopathiae
. An organism indistinguishable from
Erysipelothrix tonsillarum
was isolated for the first time from an Australian oyster and a silver bream. Overall,
Erysipelothrix
spp. were widely distributed in Australasian seafoods, illustrating the potential for erysipeloid-like infections in fishermen.
Identification of 16S Ribosomal DNA-Defined Bacterial Populations at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)
Authors: Sievert Stefan M. , Kuever Jan , Muyzer Gerard
Published: July 2002

In a recent publication (S. M. Sievert, T. Brinkhoff, G. Muyzer, W. Ziebis, and J. Kuever, Appl. Environ. Microbiol. 65:3834–3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece). Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels. A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domain
Bacteria. Cytophaga-Flavobacterium
and
Acidobacterium
were the most frequently retrieved bacterial groups. Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms. The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.
Plasmid Transfer between
Bacillus thuringiensis
subsp.
israelensis
Strains in Laboratory Culture, River Water, and Dipteran Larvae
Authors: Thomas D. John I. , Morgan J. Alun W. , Whipps John M. , Saunders Jon R.
Published: July 2002

Plasmid transfer between strains of
Bacillus thuringiensis
subsp.
israelensis
was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10
−3
transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16∷Tn
5401
was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16∷Tn
5401
was detected in water, but at a lower transfer ratio (ca. 10
−2
transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn
5401
was evident, with a maximum rate of 10
−3
transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of
B. thuringiensis
spores in the insects.
Simple Sequence Repeat Markers Distinguish among Morphotypes of
Sphaeropsis sapinea
Authors: Burgess Treena , Wingfield Michael J. , Wingfield Brenda W.
Published: July 2002

Sphaeropsis sapinea
is a fungal endophyte of
Pinus
spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of
S. sapinea
have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an
S. sapinea
isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of
S. sapinea
representing different morphotypes as well as on 2 isolates of the closely related species
Botryosphaeria obtusa
. The putative I morphotype was found to be identical to
B. obtusa
. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.
Effect of Introns and AT-Rich Sequences on Expression of the Bacterial Hygromycin B Resistance Gene in the Basidiomycete
Schizophyllum commune
Authors: Scholtmeijer Karin , Wösten Han A. B. , Springer Jan , Wessels Joseph G. H.
Published: July 2002

Previously, it was shown that introns are required for efficient mRNA accumulation in
Schizophyllum commune
and that the presence of AT-rich sequences in the coding region of genes can result in truncation of transcripts in this homobasidiomycete. Here we show that intron-dependent mRNA accumulation and truncation of transcripts are two independent events that both affect expression of the bacterial hygromycin B resistance gene in
S. commune
.
Effect of Selenite on Growth and Protein Synthesis in the Phototrophic Bacterium
Rhodobacter sphaeroides
Authors: Bebien Magali , Chauvin Jean-Paul , Adriano Jean-Marc , Grosse Sandrine , Verméglio André
Published: July 2002

The effect of selenite on the growth rate and protein synthesis has been investigated in
Rhodobacter sphaeroides
. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the
smoK
gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in
R. sphaeroides
.
Evidence for the Biosynthesis of Bryostatins by the Bacterial Symbiont “
Candidatus
Endobugula sertula” of the Bryozoan
Bugula neritina
Authors: Davidson S. K. , Allen S. W. , Lim G. E. , Anderson C. M. , Haygood M. G.
Published: July 2002

The marine bryozoan,
Bugula neritina
, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products.
B. neritina
harbors the uncultivated gamma proteobacterial symbiont “
Candidatus
Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the
B. neritina-“E. sertula”
association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total
B. neritina
DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of
B. neritina
and not to
B. neritina
cells or to other bacteria. Finally,
B. neritina
colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont
E. sertula
has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from
B. neritina
directly and use these to produce bryostatins in heterologous host bacteria.
Diversity of Toxic and Nontoxic
Nodularia
Isolates (Cyanobacteria) and Filaments from the Baltic Sea
Authors: Laamanen Maria J. , Gugger Muriel F. , Lehtimäki Jaana M. , Haukka Kaisa , Sivonen Kaarina
Published: July 2002

Cyanobacteria of the genus
Nodularia
form toxic blooms in brackish waters worldwide. In addition,
Nodularia
spp. are found in benthic, periphytic, and soil habitats. The majority of the planktic isolates produce a pentapeptide hepatotoxin nodularin. We examined the morphologic, toxicologic, and molecular characters of 18 nodularin-producing and nontoxic
Nodularia
strains to find appropriate markers for distinguishing the toxic strains from the nontoxic ones in field samples. After classical taxonomy, the examined strains were identified as
Nodularia
sp.,
Nodularia spumigena
,
N. baltica
,
N. harveyana
, and
N. sphaerocarpa
. Morphologic characters were ambiguous in terms of distinguishing between the toxic and the nontoxic strains. DNA sequences from the short 16S-23S rRNA internally transcribed spacer (ITS1-S) and from the phycocyanin operon intergenic spacer and its flanking regions (PC-IGS) were different for the toxic and the nontoxic strains. Phylogenetic analysis of the ITS1-S and PC-IGS sequences from strains identified as
N. spumigena,
and
N. baltica
, and
N. litorea
indicated that the division of the planktic
Nodularia
into the three species is not supported by the ITS1-S and PC-IGS sequences. However, the ITS1-S and PC-IGS sequences supported the separation of strains designated
N. harveyana
and
N. sphaerocarpa
from one another and the planktic strains.
Hae
III digestion of PCR amplified PC-IGS regions of all examined 186
Nodularia
filaments collected from the Baltic Sea produced a digestion pattern similar to that found in toxic isolates. Our results suggest that only one planktic
Nodularia
species is present in the Baltic Sea plankton and that it is nodularin producing.
Genetic and Functional Analysis of the
tbc
Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in
Burkholderia
sp. Strain JS150
Authors: Kahng Hyung-Yeel , Malinverni Juliana C. , Majko Michelle M. , Kukor Jerome J.
Published: July 2002

Burkholderia
sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated
tbc
(for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in
Pseudomonas aeruginosa
PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The
tbc
genes are organized into two divergently transcribed operons,
tbc1
and
tbc2
, each comprised of six open reading frames. Similarity searches of databases revealed that the
tbc1
and
tbc2
genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that
tbc2
plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the
tbc1
gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these
tbc
monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.
Expression of a
Pseudomonas putida
Aminotransferase Involved in Lysine Catabolism Is Induced in the Rhizosphere
Authors: Espinosa-Urgel Manuel , Ramos Juan-Luis
Published: July 2002

Using a transposon carrying a promoterless
lux
operon to generate transcriptional fusions by insertional mutagenesis, we have identified a
Pseudomonas putida
gene with increased expression in the presence of corn root exudates. Expression of the transcriptional fusion, induced by the amino acid lysine, was detected in
P
.
putida
in the rhizosphere of plants as well as in response to seed exudates. The mutant was unable to grow on lysine or δ-aminovalerate as carbon sources, which indicates that the affected function is involved in the pathway for lysine catabolism. However, the mutant strain grew with glutaric acid, the product of δ-aminovalerate metabolism via glutaric acid semialdehyde, as a C source. The translated sequence of the interrupted gene showed high levels of similarity with aminotransferases. These sets of data suggest that the product of this gene has δ-aminovalerate aminotransferase activity. This is the first direct genetic evidence correlating a DNA sequence with such activity in
Pseudomonadaceae
.
Cyt1Aa from
Bacillus thuringiensis
subsp.
israelensis
Is Toxic to the Diamondback Moth,
Plutella xylostella
, and Synergizes the Activity of Cry1Ac towards a Resistant Strain
Authors: Sayyed Ali H. , Crickmore Neil , Wright Denis J.
Published: July 2002

The
Bacillus thuringiensis
subsp.
israelensis
cytolytic protein Cyt1Aa was found to be toxic to an insecticide-susceptible laboratory population of
Plutella xylostella
. Cry1Ac-resistant populations of
P. xylostella
showed various degrees of resistance to Cyt1Aa. Cyt1Aa/Cry1Ac mixtures showed a marked level of synergism in the Cry1Ac-resistant populations.
Characterization of Rhizobial Isolates of
Phaseolus vulgaris
by Staircase Electrophoresis of Low-Molecular-Weight RNA
Authors: Velázquez Encarna , Martı́nez-Romero Esperanza , Rodrı́guez-Navarro Dulce Nombre , Trujillo Martha E. , Daza Antonio , Mateos Pedro F. , Martı́nez-Molina Eustoquio , van Berkum Peter
Published: July 2002

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as
Rhizobium etli
,
Rhizobium gallicum
,
Rhizobium giardinii
,
Rhizobium leguminosarum
bv. viciae and bv. trifolii, and
Sinorhizobium fredii
.
Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of
Xylella fastidiosa
Authors: Hendson Mavis , Purcell Alexander H. , Chen Deqiao , Smart Chris , Guilhabert Magalie , Kirkpatrick Bruce
Published: July 2002

Strains of
Xylella fastidiosa
isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products,
Not
I and
Spe
I genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of
X. fastidiosa
isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of
X. fastidiosa
at the subspecies or pathovar level.
Community Structure of Denitrifiers,
Bacteria
, and
Archaea
along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (
nirS
) and 16S rRNA Genes
Authors: Braker Gesche , Ayala-del-Rı́o Héctor L. , Devol Allan H. , Fesefeldt Andreas , Tiedje James M.
Published: July 2002

Steep vertical gradients of oxidants (O
2
and NO
3
−
) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers,
Bacteria
, and
Archaea
along redox gradients at distant geographic locations, nitrite reductase (
nirS
) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of
nirS
-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of
nirS
to computer-simulated T-RFs of
nirS
clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of
nirS
and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of
Archaea
was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.
Cloning and Expression of a
Ralstonia eutropha
HF39 Gene Mediating Indigo Formation in
Escherichia coli
Authors: Drewlo Sascha , Brämer Christian O. , Madkour Mohamed , Mayer Frank , Steinbüchel Alexander
Published: July 2002

On complex medium
Escherichia coli
strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of
Ralstonia eutropha
HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (
bec
) was cloned and sequenced. The deduced amino acid sequence of
bec
showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.
New Pulp Biobleaching System Involving Manganese Peroxidase Immobilized in a Silica Support with Controlled Pore Sizes
Authors: Sasaki Toshiya , Kajino Tsutomu , Li Bo , Sugiyama Hidehiko , Takahashi Haruo
Published: July 2002

Attempts have been made to use manganese peroxidase (MnP) for chlorine-free pulp biobleaching, but they have not been commercially viable because of the enzyme's low stability. We developed a new pulp biobleaching method involving mesoporous material-immobilized manganese peroxidase from
Phanerochaete chrysosporium
. MnP immobilized in FSM-16, a folded-sheet mesoporous material whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability and tolerance to H
2
O
2
. MnP immobilized in FSM-16 retained more than 80% of its initial activity even after 10 days of continuous reaction. We constructed a thermally discontinuous two-stage reactor system, in which the enzyme (39°C) and pulp-bleaching (70°C) reactions were performed separately. When the treatment of pulp with MnP by means of the two-stage reactor system and alkaline extraction was repeated seven times, the brightness of the pulp increased to about 88% within 7 h after completion of the last treatment.
Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques
Authors: Dı́ez Beatriz , Pedrós-Alió Carlos , Marsh Terence L. , Massana Ramon
Published: July 2002

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.
Detection and Identification of Mammalian Reoviruses in Surface Water by Combined Cell Culture and Reverse Transcription-PCR
Authors: Spinner Michael L. , Di Giovanni George D.
Published: July 2002

Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture–reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the λ3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.
Killer Toxin ofKluyveromyces phaffiiDBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts
Authors: Ciani M. , Fatichenti F.
Published: July 2002

The use ofKluyveromyces phaffiiDBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin ofK. phaffiiDBVPG 6076 showed extensive anti-Hanseniasporaactivity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40°C. These biochemical properties would allow the use ofK. phaffiikiller toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain ofHanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin toH. uvarumcells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that theK. phaffiiDBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.
Organization and Regulation of
meta
Cleavage Pathway Genes for Toluene and
o
-Xylene Derivative Degradation in
Pseudomonas stutzeri
OX1
Authors: Arenghi Fabio L. G. , Berlanda Davide , Galli Enrica , Sello Guido , Barbieri Paola
Published: July 2002

Pseudomonas stutzeri
OX1
meta
pathway genes for toluene and
o
-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the
Pseudomonas
sp. strain CF600
dmp
operon for phenol catabolism and was found to be coregulated by the
tou
operon activator TouR. A hypothesis about the evolution of the toluene and
o
-xylene catabolic pathway in
P. stutzeri
OX1 is discussed.
The
ybiT
Gene of
Erwinia chrysanthemi
Codes for a Putative ABC Transporter and Is Involved in Competitiveness against Endophytic Bacteria during Infection
Authors: Llama-Palacios Arancha , López-Solanilla Emilia , Rodríguez-Palenzuela Pablo
Published: July 2002

We investigated the role in bacterial infection of a putative ABC transporter, designated
ybiT
, of
Erwinia chrysanthemi
AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as
Escherichia coli
and
Pseudomonas aeruginosa
, as well as structural similarity with proteins of
Streptomyces
spp. involved in resistance to macrolide antibiotics. The gene contiguous to
ybiT
, designated as
pab
(putative antibiotic biosynthesis) showed sequence similarity with
Pseudomonas
and
Streptomyces
genes involved in the biosynthesis of antibiotics. A
ybiT
mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the
ybiT
gene plays a role in the in planta fitness of the bacteria.
Denaturation of Either
Manduca sexta
Aminopeptidase N or
Bacillus thuringiensis
Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions
Authors: Daniel Anu , Sangadala Sreedhara , Dean Donald H. , Adang Michael J.
Published: July 2002

The effect of polypeptide denaturation of
Bacillus thuringiensis
Cry1A toxins or purified
Manduca sexta
120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with
125
I-labeled Cry1Ac, Cry1Ac mutant
509
QNR-AAA
511
(QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the
N
-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both
125
I-Cry1Ac and
125
I-QNR-AAA bound to 210- and 120-kDa proteins from
M. sexta
brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots
125
I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment,
125
I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA.
125
I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured
125
I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured)
125
I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.
Molecular Characterization of the Leucine Cluster in
Buchnera
sp. Strain PSY, a Primary Endosymbiont of the Aphid
Pemphigus spyrothecae
Authors: Sabater-Muñoz Beatriz , Gómez-Valero Laura , van Ham Roeland C. H. J. , Silva Francisco J. , Latorre Amparo
Published: July 2002

Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (
leuA
, -
B
, -
C
, and -
D
) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in
Buchnera
sp. strain PSY from the aphid
Pemphigus spyrothecae
(subfamily Pemphiginae). The genes that flank
leuABCD
in
Buchnera
sp. strain PSY appear to be adjacent in the genome of
Buchnera
sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic
Buchnera
and that the chromosomal location observed in
Buchnera
sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome.
Antibody to Heat Shock Protein Can Be Used for Early Serological Monitoring of
Helicobacter pylori
Eradication Treatment
Authors: Yunoki Naoko , Yokota Kenji , Mizuno Motowo , Kawahara Yoshiro , Adachi Masayasu , Okada Hiroyuki , Hayashi Shyunji , Hirai Yoshikazu , Oguma Keiji , Tsuji Takao
Published: July 2002

Infection with
Helicobacter pylori
induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including
H. pylori
infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with
H. pylori
-infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the
H. pylori
hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of
H. pylori
hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to
H. pylori
hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.
Comparison of Five Serologic Tests for Diagnosis of Acute Infections by
Chlamydia pneumoniae
Authors: Persson Kenneth , Boman Jens
Published: July 2002

Serology is often used to diagnose acute infections by
Chlamydia pneumoniae
. In this study paired sera from patients with acute respiratory tract infection during an epidemic of
C. pneumoniae
infections were examined by five different antibody tests. These tests were the complement fixation (CF) test, the microimmunofluorescence (MIF) test, a recombinant enzyme immunoassay (rEIA) (Medac) based on a recombinant lipopolysaccharide of chlamydia and measuring antibodies to a common chlamydial antigen, and two tests that utilize preparations of
C. pneumoniae
organisms, the SeroCp-EIA (Savyon) (with preserved lipopolysaccharide) and the LOY-EIA (Labsystems) (without this antigen). Both of the last two tests should measure specific antibodies to
C. pneumoniae
, although cross-reacting antibodies may also be detected by the SeroCp-EIA. Acute infection of
C. pneumoniae
was serologically confirmed in 44% of the cases by at least two different tests. Using an expanded “gold standard,” i.e., the presence of significant reactions in at least two tests, the sensitivity of the CF test was 69%, that of the MIF test was 88%, that of the rEIA was 89%, that of the LOY-EIA was 96%, and that of the SeroCp-EIA was 92%. Specificity was high for all methods, but adjustments of diagnostic criteria were made to several of the tests. The basis for these adjustments and supportive data are presented. Infections of
C. pneumoniae
were detected in patients from 8 to 83 years of age. Two peaks in the incidence of such infections were observed: one among young teenagers and a second in adults 30 to 45 years of age, corresponding to parents of young teen-agers. The tests were equally sensitive in different age groups. Reinfections seemed to be rare.
Comparison of a Classical Phagocytosis Assay and a Flow Cytometry Assay for Assessment of the Phagocytic Capacity of Sera from Adults Vaccinated with a Pneumococcal Conjugate Vaccine
Authors: Jansen Wouter T. M. , Väkeväinen-Anttila Merja , Käyhty Helena , Nahm Moon , Bakker N. , Verhoef Jan , Snippe Harm , Verheul André F. M.
Published: July 2002

Antibody- and complement-mediated phagocytosis is the main defense mechanism against
Streptococcus pneumoniae
. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.
Influence of Patient Age on
Streptococcus pneumoniae
Serotypes Causing Invasive Disease
Authors: Inostroza Jaime , Vinet Ana Maria , Retamal Gloria , Lorca Pedro , Ossa Gonzalo , Facklam Richard R. , Sorensen Ricardo U.
Published: July 2002

All clinical
S. pneumoniae
specimens isolated from patients with invasive or sterile-site infections admitted to one regional general hospital in southern Chile were collected during a 5-year period (February 1994 to September 1999). A total of 247 strains belonging to 50 serotypes were isolated in this survey: 69 in patients under 5 years of age, 129 in patients 5 to 64 years old, and 49 from patients 65 years and older. Eight serotypes were identified in all age groups, while all other serotypes were found exclusively in one age group or in patients over 4 years of age. Serotype 3 was never found in patients under 5 years old, and serotype 14 was not found in patients >64 years of age. There was no difference in the serotypes causing infection in each one of the 5 years of the survey. Our results suggest that both bacterial virulence factors and host factors play an important role in the selection of
S. pneumoniae
serotypes causing invasive infection. Possible host factors include age-related differences in the immune response. Comparative studies with other areas of the world may help to further understanding of our observations in southern Chile.