bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of an Isolate That Uses Vinyl Chloride as a Growth Substrate under Aerobic Conditions
Authors: Verce Matthew F. , Ulrich Ricky L. , Freedman David L.
Published: July 2002

An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene as
Pseudomonas aeruginosa
, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 μmol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 μM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.
Heterologous Coproduction of Enterocin A and Pediocin PA-1 by
Lactococcus lactis
: Detection by Specific Peptide-Directed Antibodies
Authors: Martínez José M. , Kok Jan , Sanders Jan W. , Hernández Pablo E.
Published: July 2002

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in
Lactococcus lactis
IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martı́nez, M. I. Martı́nez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodrı́guez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536–4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by
L. lactis
IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.
Germination-Induced Bioluminescence, a Route To Determine the Inhibitory Effect of a Combination Preservation Treatment on Bacterial Spores
Authors: Ciarciaglini Gianni , Hill Philip J. , Davies Ken , McClure Peter J. , Kilsby Derrick , Brown Martyn H. , Coote Peter J.
Published: July 2002

In this work, we have used spores of
Bacillus subtilis
that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0.5%), and a pasteurization step (90°C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the
l
-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on
l
-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.
Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass,
Spartina alterniflora
Authors: Lovell Charles R. , Piceno Yvette M. , Quattro Joseph M. , Bagwell Christopher E.
Published: July 2002

N
2
fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass,
Spartina alterniflora
, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified
nifH
sequence segments was used in previous studies to examine the stability and dynamics of the
Spartina
rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the
nifH
amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the
Spartina
rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division
Proteobacteria
(γ-
Proteobacteria
) and from various anaerobic diazotrophs. Diazotrophs in the α-
Proteobacteria
were poorly represented. None of the
Spartina
rhizosphere DGGE band sequences were identical to any known or previously recovered environmental
nifH
sequences. The
Spartina
rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.
The Group I Strain of
Streptococcus mutans
, UA140, Produces Both the Lantibiotic Mutacin I and a Nonlantibiotic Bacteriocin, Mutacin IV
Authors: Qi Fengxia , Chen Ping , Caufield Page W.
Published: July 2002

Strains of
Streptococcus mutans
produce at least three mutacins, I, II, and III. Mutacin II is a member of subgroup AII in the lantibiotic family of bacteriocins, and mutacins I and III belong to subgroup AI in the lantibiotic family. In this report, we characterize two mutacins produced by UA140, a group I strain of
S. mutans
. One is identical to the lantibiotic mutacin I produced by strain CH43 (F. Qi et al., Appl. Environ. Microbiol. 66:3221–3229, 2000); the other is a nonlantibiotic bacteriocin, which we named mutacin IV. Mutacin IV belongs to the two-peptide, nonlantibiotic family of bacteriocins produced by gram-positive bacteria. Peptide A, encoded by gene
nlmA
, is 44 amino acids (aa) in size and has a molecular mass of 4,169 Da; peptide B, encoded by
nlmB
, is 49 aa in size and has a molecular mass of 4,826 Da. Both peptides derive from prepeptides with glycines at positions −2 and −1 relative to the processing site. Production of mutacins I and IV by UA140 appears to be regulated by different mechanisms under different physiological conditions. The significance of producing two mutacins by one strain under different conditions and the implication of this property in terms of the ecology of
S. mutans
in the oral cavity are discussed.
Genetic and Molecular Organization of the Alkylbenzene Catabolism Operon in the Psychrotrophic Strain
Pseudomonas putida
01G3
Authors: Chablain P. A. , Zgoda A. L. , Sarde C.-O. , Truffaut N.
Published: July 2002

The 11-kb sequence encompassing the alkylbenzene upper pathway in
Pseudomonas putida
01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in
P. putida
RE204.
ebd
genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS
5
-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster.
Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation
Authors: Futamata Hiroyuki , Harayama Shigeaki , Watanabe Kazuya
Published: July 2002

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-
K

s

(the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-
K

s

, and high-
K

s

groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter
−1
); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of
Methylosinus trichosporium
OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.
Fungal Growth, Production, and Sporulation during Leaf Decomposition in Two Streams
Authors: Suberkropp Keller
Published: July 2002

I examined the activity of fungi associated with yellow poplar (
Liriodendron tulipifera
) and white oak (
Quercus alba
) leaves in two streams that differed in pH and alkalinity (a hardwater stream [pH 8.0] and a softwater stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 μg liter
−1
) and phosphorus (<3 μg liter
−1
). The leaves of each species decomposed faster in the hardwater stream (decomposition rates, 0.010 and 0.007 day
−1
for yellow poplar and oak, respectively) than in the softwater stream (decomposition rates, 0.005 and 0.004 day
−1
for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [
14
C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hardwater stream but not in the softwater stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hardwater stream (5.8 mg g
−1
day
−1
on yellow poplar leaves and 3.1 mg g
−1
day
−1
on oak leaves) than in the softwater stream (1.6 mg g
−1
day
−1
on yellow poplar leaves and 0.9 mg g
−1
day
−1
on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the softwater stream (12.8% of detrital mass) than in the hardwater stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the softwater stream.
Direct Detection by In Situ PCR of the
amoA
Gene in Biofilm Resulting from a Nitrogen Removal Process
Authors: Hoshino Tatsuhiko , Noda Naohiro , Tsuneda Satoshi , Hirata Akira , Inamori Yuhei
Published: July 2002

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the
amoA
gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the
amoA
gene present in
Nitrosomonas europaea
cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The
amoA
gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the
amoA
gene in the biofilms was detected without a nonspecific signal, and the cells possessing the
amoA
gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.
Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization
Authors: Daims Holger , Ramsing Niels B. , Schleifer Karl-Heinz , Wagner Michael
Published: July 2002

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of
Escherichia coli
cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 10
7
± 1.9 × 10
7
cells ml
−1
. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.
Molecular Characterization of the Iron-Hydroxamate Uptake System in
Staphylococcus aureus
Authors: Cabrera Guillermo , Xiong Anming , Uebel Michelle , Singh Vineet K. , Jayaswal Radheshyam K.
Published: July 2002

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated
fhuC
,
fhuB
, and
fhuD
, was identified in
Staphylococcus aureus
. Whereas the
fhuC
gene encodes an ATP-binding protein,
fhuB
and
fhuD
code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A
fhuB
knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.
Binding Analyses of
Bacillus thuringiensis
Cry δ-Endotoxins Using Brush Border Membrane Vesicles of
Ostrinia nubilalis
Authors: Hua Gang , Masson Luke , Jurat-Fuentes Juan Luis , Schwab George , Adang Michael J.
Published: July 2002

Transgenic corn expressing the
Bacillus thuringiensis
Cry1Ab gene is highly insecticidal to
Ostrinia nubilalis
(European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the
O. nubilalis
brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured
125
I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for
125
I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit
125
I-Cry1Ab binding to BBMV. Cry1F inhibited
125
I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-
Manduca sexta
aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.
Archaeal Diversity in Waters from Deep South African Gold Mines
Authors: Takai Ken , Moser Duane P. , DeFlaun Mary , Onstott Tullis C. , Fredrickson James K.
Published: July 2002

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the
Crenarchaeota
. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of
Euryarchaeota
. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain
Archaea
are reevaluated.
Microbial Populations Involved in Cycling of Dimethyl Sulfide and Methanethiol in Freshwater Sediments
Authors: Lomans Bart P. , Luderer Rianne , Steenbakkers Peter , Pol Arjan , van der Drift Chris , Vogels Godfried D. , Op den Camp Huub J. M.
Published: July 2002

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related toMethanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene fromM. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogenM. hollandicain freshwater sediments.
Emulsifying Activities of Purified Alasan Proteins from
Acinetobacter radioresistens
KA53
Authors: Toren Amir , Navon-Venezia Shiri , Ron Eliora Z. , Rosenberg Eugene
Published: July 2002

The bioemulsifier of
Acinetobacter radioresistens
KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. The emulsifying activity of the purified polysaccharide (apo-alasan) is very low. Three of the alasan proteins were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins demonstrated that the active components of the alasan complex are the proteins. The 45-kDa protein had the highest specific emulsifying activity, 11% higher than the intact alasan complex. The 16- and 31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a 1.8-fold increase in the specific emulsifying activity and increased stability of the oil-in-water emulsion. Fast-performance liquid chromatography analysis indicated that the 45-kDa protein forms a dimer in nondenaturing conditions and interacts with the 16- and 31-kDa proteins to form a high-molecular-mass complex. The 45-kDa protein and the three-protein complex had substrate specificities for emulsification and a range of pH activities similar to that of alasan. The fact that the purified proteins are active emulsifiers should simplify structure-function studies and advance our understanding of their biological roles.
Cryptosporidium parvum
Infection Involving Novel Genotypes in Wildlife from Lower New York State
Authors: Perz Joseph F. , Le Blancq Sylvie M.
Published: July 2002

Cryptosporidium
, an enteric parasite of humans and a wide range of other mammals, presents numerous challenges to the supply of safe drinking water. We performed a wildlife survey, focusing on white-tailed deer and small mammals, to assess whether they may serve as environmental sources of
Cryptosporidium
. A PCR-based approach that permitted genetic characterization via sequence analysis was applied to wildlife fecal samples (
n
= 111) collected from September 1996 to July 1998 from three areas in lower New York State. Southern analysis revealed 22 fecal samples containing
Cryptosporidium
small-subunit (SSU) ribosomal DNA; these included 10 of 91 white-tailed deer (
Odocoileus virginianus
) samples, 3 of 5 chipmunk (
Tamias striatus
) samples, 1 of 2 white-footed mouse (
Peromyscus leucopus
) samples, 1 of 2 striped skunk (
Mephitis mephitis
) samples, 1 of 5 racoon (
Procyon lotor
) samples, and 6 of 6 muskrat (
Ondatra zibethicus
) samples. All of the 15 SSU PCR products sequenced were characterized as
Cryptosporidium parvum
; two were identical to genotype 2 (bovine), whereas the remainder belonged to two novel SSU sequence groups, designated genotypes 3 and 4. Genotype 3 comprised four deer-derived sequences, whereas genotype 4 included nine sequences from deer, mouse, chipmunk, and muskrat samples. PCR analysis was performed on the SSU-positive fecal samples for three other
Cryptosporidium
loci (dihydrofolate reductase, polythreonine-rich protein, and beta-tubulin), and 8 of 10 cloned PCR products were consistent with
C. parvum
genotype 2. These data provide evidence that there is sylvatic transmission of
C. parvum
involving deer and other small mammals. This study affirmed the importance of wildlife as potential sources of
Cryptosporidium
in the catchments of public water supplies.
Spiroplasma
Symbiont of the Pea Aphid,
Acyrthosiphon pisum
(Insecta: Homoptera)
Authors: Fukatsu Takema , Tsuchida Tsutomu , Nikoh Naruo , Koga Ryuichi
Published: July 2002

From a laboratory strain of the pea aphid,
Acyrthosiphon pisum,
we discovered a previously unknown facultative endosymbiotic bacterium. Molecular phylogenetic analysis based on 16S ribosomal DNA revealed that the bacterium is a member of the genus
Spiroplasma
. The
Spiroplasma
organism showed stable vertical transmission through successive generations of the host. Injection of hemolymph from infected insects into uninfected insects established a stable infection in the recipients. The
Spiroplasma
symbiont exhibited negative effects on growth, reproduction, and longevity of the host, particularly in older adults. Of 58 clonal strains of
A. pisum
established from natural populations in central Japan, 4 strains possessed the
Spiroplasma
organism.
Determination of Virus Abundance in Marine Sediments
Authors: Danovaro R. , Dell'Anno A. , Trucco A. , Serresi M. , Vanucci S.
Published: July 2002

In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.
Quantification of Trichothecene-Producing
Fusarium
Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter Wheat
Authors: Edwards S. G. , Pirgozliev S. R. , Hare M. C. , Jenkinson P.
Published: July 2002

We developed a PCR-based assay to quantify trichothecene-producing
Fusarium
based on primers derived from the trichodiene synthase gene (
Tri
5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing
Fusarium
species. Amounts of the trichothecene-producing
Fusarium
and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing
Fusarium
and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing
Fusarium
compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing
Fusarium
in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing
Fusarium
within the FHB disease complex and not by altering the rate of DON production. The
Tri
5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.
Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by
Pseudomonas syringae
Strains and Their Potential Use in Strain Identification
Authors: Bultreys Alain , Gheysen Isabelle , Maraite Henri , de Hoffmann Edmond
Published: July 2002

Nonfluorescent highly virulent strains of
Pseudomonas syringae
pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent
P. syringae
strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent
P. syringae
strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent
Pseudomonas
species and fluorescent
P. syringae
and
P. viridiflava
strains and to distinguish between the two siderovars in
P. syringae
pv. aptata.
Induction of Laccase Activity in
Rhizoctonia solani
by Antagonistic
Pseudomonas fluorescens
Strains and a Range of Chemical Treatments
Authors: Crowe Jonathan D. , Olsson Stefan
Published: July 2002

Fungi often produce the phenoloxidase enzyme laccase during interactions with other organisms, an observation relevant to the development of biocontrols. By incorporating the laccase substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar, we analyzed laccase induction in the plant-pathogenic fungus
Rhizoctonia solani
when paired against isolates of the soil bacterium
Pseudomonas fluorescens
. Substantial induction of
R. solani
laccase was seen only in pairings with strains of
P. fluorescens
known to produce antifungal metabolites. To study laccase induction further, a range of chemical treatments was applied to
R. solani
liquid cultures.
p
-Anisidine, copper(II), manganese(II), calcium ionophore
A23187
, lithium chloride, calcium chloride, cyclic AMP (cAMP), caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all found to induce laccase; however, the
P. fluorescens
metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes in lipid peroxidation levels and dry weight. The results indicated that the laccase induction seen in pairing plate experiments was most likely due to calcium or heat shock signaling in response to the effects of bacterial metabolites, but that heavy metal and cAMP-driven laccase induction was involved in sclerotization.
Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish
Authors: Nikoskelainen Sami , Salminen Seppo , Bylund Göran , Ouwehand Arthur C.
Published: July 2002

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use,
Lactobacillus rhamnosus
ATCC 53103,
Lactobacillus casei
Shirota,
Lactobacillus bulgaricus, L. rhamnosus
LC 705,
Bifidobacterium lactis
Bb12, and
Lactobacillus johnsonii
La1, and one for animal use,
Enterococcus faecium
Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens,
Vibrio anguillarum, Aeromonas salmonicida
, and
Flavobacterium psychrophilum
, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of
A. salmonicida
. Coculture experiments showed significant inhibition of growth of
A. salmonicida
, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except
L. casei
Shirota showed tolerance against fish bile.
L. rhamnosus
ATCC 53103 and
L. bulgaricus
were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth,
L. rhamnosus
ATCC 53103 and
L. bulgaricus
can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.
Kinetics of Perchlorate- and Chlorate-Respiring Bacteria
Authors: Logan Bruce E. , Zhang Husen , Mulvaney Peter , Milner Michael G. , Head Ian M. , Unz Richard F.
Published: July 2002

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “
Dechlorosoma
” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h
−1
, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h
−1
, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h
−1
, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h
−1
, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h
−1
) and exceeded that of strain KJ on perchlorate (0.14 h
−1
). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [
n
= 7]), chlorate (0.44 ± 0.05 [
n
= 7]), and perchlorate (0.50 ± 0.08 [
n
= 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.
Purification, Characterization, and Functional Role of a Novel Extracellular Protease from
Pleurotus ostreatus
Authors: Palmieri Gianna , Bianco Carmen , Cennamo Giovanna , Giardina Paola , Marino Gennaro , Monti Maria , Sannia Giovanni
Published: July 2002

A new extracellular protease (PoSl;
Pleurotus ostreatus
subtilisin-like protease) from
P. ostreatus
culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the
K

m

value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca
2+
binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of
P. ostreatus
laccase activity by degrading and/or activating different isoenzymes.
Burkholderia
, a Genus Rich in Plant-Associated Nitrogen Fixers with Wide Environmental and Geographic Distribution
Authors: Estrada-De Los Santos Paulina , Bustillos-Cristales Rocı́o , Caballero-Mellado Jesús
Published: July 2002

The genus
Burkholderia
comprises 19 species, including
Burkholderia vietnamiensis
which is the only known N
2
-fixing species of this bacterial genus. The first isolates of
B. vietnamiensis
were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N
2
-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as
B. cepacia
using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of
B. vietnamiensis
TVV75
T
. These features include nitrogenase activity with 10 different carbon sources, identical or very similar
nifHDK
hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75
T
. Although the ability to fix N
2
is not reported to be a common feature among the known species of the genus
Burkholderia
, the results obtained show that many diazotrophic
Burkholderia
isolates analyzed showed phenotypic and genotypic features different from those of the known N
2
-fixing species
B. vietnamiensis
as well as from those of
B. kururiensis
, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N
2
-fixing
Burkholderia
species different from
B. vietnamiensis
. In addition, this study shows the wide geographic distribution and substantial capability of N
2
-fixing
Burkholderia
spp. for colonizing diverse host plants in distantly separated environments.
Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker
Authors: Cubero J. , Graham J. H. , Gottwald T. R.
Published: July 2002

For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions.
Effect of High-Rate Algal Ponds on Viability of
Cryptosporidium parvum
Oocysts
Authors: Araki S. , Martı́n-Gomez S. , Bécares E. , De Luis-Calabuig E. , Rojo-Vazquez F.
Published: July 2002

The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction in the infectivity of
Cryptosporidium parvum
oocysts in neonatal mice. The use of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater, supporting the importance of alga-based systems for safer reuse of treated wastewater.
Contamination of River Water by
Cryptosporidium parvum
Oocysts in Western Japan
Authors: Ono Kazuo , Tsuji Hidetaka , Rai Shiba Kumar , Yamamoto Akio , Masuda Kuniyoshi , Endo Takuro , Hotta Hak , Kawamura Takashi , Uga Shoji
Published: July 2002

In Japan, only a few rivers have been inspected for
Cryptosporidium parvum
contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for
Cryptosporidium
oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for
Cryptosporidium
oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the
C. parvum
found as the bovine or human type.
C. parvum
was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained
C. parvum
oocysts. The number of
C. parvum
oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the
C. parvum
detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with
C. parvum
oocysts, as reported in other countries.
Low-Temperature Lipase from Psychrotrophic
Pseudomonas
sp. Strain KB700A
Authors: Rashid Naeem , Shimada Yuji , Ezaki Satoshi , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2002

We have previously reported that a psychrotrophic bacterium,
Pseudomonas
sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into
Escherichia coli
TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (
KB-lip
) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from
Pseudomonas fluorescens
and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca
2+
. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca
2+
-dependent lipase. Addition of Mn
2+
and Sr
2+
also led to enhancement of lipase activity but to a much lower extent than that produced by Ca
2+
. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When
p
-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C
10
acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.