bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from
Arthrobacter
sp. Strain SU
Authors: Zhuang Zhihao , Gartemann Karl-Heinz , Eichenlaub Rudolf , Dunaway-Mariano Debra
Published: May 2003

The
Arthrobacter
sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes
fcbA
,
fcbB
, and
fcbC
(A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes
fcbA
and
fcbB
encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of
fcbC
is not known. We subcloned
fcbC
and expressed it in
Escherichia coli
, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a
k
cat
of 6.7 s
−1
and a

K
m

of 1.2 μM. The
k
cat
pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading
Pseudomonas
strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.
Characterization of Fe(III) Reduction by Chlororespiring
Anaeromxyobacter dehalogenans
Authors: He Qiang , Sanford Robert A.
Published: May 2003

Anaeromyxobacter dehalogenans
strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of
ortho
-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [
14
C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.
Electrophoretic Analysis of Genetic Variability within
Cryptosporidium parvum
from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom
Authors: Gasser R. B. , EL-Osta Y. G. Abs , Chalmers R. M.
Published: May 2003

Cryptosporidium parvum
oocyst DNA samples (
n
= 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (∼300 bp) and second internal transcribed spacer (pITS-2) (∼230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of
C. parvum
could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of ∼1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as
C. parvum
type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (
n
= 20; one child nursery outbreak) and type 2 (
n
= 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of ∼1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.
Identification of New Genes Positively Regulated by
Tri10
and a Regulatory Network for Trichothecene Mycotoxin Production
Authors: Peplow Andrew W. , Tag Andrew G. , Garifullina Gulnara F. , Beremand Marian N.
Published: May 2003

Tri10
, a regulatory gene in trichothecene mycotoxin-producing
Fusarium
species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (
Tri4
,
Tri5
,
Tri6
, and
Tri101
) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (
FPPS
). We showed that six more trichothecene genes (
Tri3
,
Tri7
,
Tri8
,
Tri9
,
Tri11
, and
Tri12
) are regulated by
Tri10
. We also constructed a cDNA library from a strain of
Fusarium sporotrichioides
that overexpresses
Tri10
(↑
Tri10
) and used cDNA derived from the ↑
Tri10
strain and a non-
Tri10
-expressing strain (Δ
Tri10
) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional
Tri10
-regulated transcripts. Four of these transcripts represent
Tri1
,
Tri13
, and
Tri14
and a gene designated
Tri15
. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated
Ibt
(influenced by
Tri10
) genes. Of the 26 transcripts now known to be positively regulated by
Tri10
, 22 are positively coregulated by
Tri6
, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22
Tri10
- and
Tri6
-coregulated sequences include all of the known
Tri
genes (except for
Tri10
), the
FPPS
gene, and the other three putative isoprenoid biosynthetic genes.
Tri6
also regulates a transcript that is not regulated by
Tri10
. Thus,
Tri10
and
Tri6
regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.
Purification and Characterization of Streptin, a Type A1 Lantibiotic Produced by
Streptococcus pyogenes
Authors: Wescombe Philip A. , Tagg John R.
Published: May 2003

Approximately 10% of
Streptococcus pyogenes
strains inhibit the growth of all nine indicators in a standardized streptococcal bacteriocin typing scheme. The present study has shown that this inhibitory profile, referred to as bacteriocin producer (P)-type 777 activity, is due to the type A1 lantibiotic streptin. Two major forms of streptin were purified to homogeneity from 95% acidified (pH 2) methanol extracts of
S. pyogenes
M25 cells by using a series of reversed-phase chromatographic separations. The fully processed form of streptin (streptin 1) is a 23-amino-acid peptide with a mass of 2,424 Da. The 2,821-
M
r
form of the peptide (streptin 2) has three additional amino acids (TPY) at the N terminus. Strain M25 extracts also contained small quantities of the streptin 1 and streptin 2 peptides in various stages of dehydration. Streptin 1 and streptin 2 were each capable of specifically inducing streptin production when added to strain M25 cultures. The streptin gene cluster resembled that of other type A1 lantibiotics but appeared to lack a streptin-specific proteinase gene. Although the streptin structural gene (
srtA
) was widespread within
S. pyogenes
, being detected in 40 of 58 strains, each representing a different M serotype, only 10 of these
srtA
-positive strains produced active streptin. The failure of some strains to express streptin was attributed to an ∼4.5-kb deletion in their streptin loci, encompassing genes putatively encoding proteins involved in streptin processing (
srtB
and
srtC
) and transport (
srtT
). In other strains,
srtA
transcription appeared to be defective. No direct association could be detected between the production of streptin and the production of the lantibiotic-like hemolysin streptolysin S in strain M25.
Homologous
npdGI
Genes in 2,4-Dinitrophenol- and 4-Nitrophenol-Degrading
Rhodococcus
spp
Authors: Heiss Gesche , Trachtmann Natalie , Abe Yoshikatsu , Takeo Masahiro , Knackmuss Hans-Joachim
Published: May 2003

Rhodococcus
(
opacus
)
erythropolis
HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F
420
reductase (NDFR; encoded by
npdG
) and the hydride transferase II (HTII; encoded by
npdI
) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study,
npdG
and
npdI
were amplified from six 2,4-DNP degrading
Rhodococcus
spp. The genes showed sequence similarities of 86 to 99% to the respective
npd
genes of strain HL PM-1. Heterologous expression of the
npdG
and
npdI
genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F
420
. Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F
420
-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F
420
-dependent glucose-6-phosphate dehydrogenases and F
420
-dependent
N
5
,
N
10
-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F
420
-dependent enzymes involved in catabolism.
Genome Differences That Distinguish
Bacillus anthracis
from
Bacillus cereus
and
Bacillus thuringiensis
Authors: Radnedge Lyndsay , Agron Peter G. , Hill Karen K. , Jackson Paul J. , Ticknor Lawrence O. , Keim Paul , Andersen Gary L.
Published: May 2003

The three species of the group 1 bacilli,
Bacillus anthracis
,
B. cereus
, and
B. thuringiensis
, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes.
B. anthracis
is the causative agent of anthrax and is phylogenetically monomorphic, while
B. cereus
and
B. thuringiensis
are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing
Bacillus
species, some of which show significant similarity to
B. anthracis
. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from
B. anthracis
Ames. Ninety-three DNA sequences that were present in
B. anthracis
but absent from the non-anthrax-causing
Bacillus
genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing
Bacillus
species but were present in all members of a representative collection of
B. anthracis
strains. These sequences map to distinct loci on the
B. anthracis
genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of
B. anthracis
.
Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin
Authors: Dhillon Ashita , Teske Andreas , Dillon Jesse , Stahl David A. , Sogin Mitchell L.
Published: May 2003

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (
dsrAB
) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The
dsrAB
sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus
Desulfobacter
and a clade of novel, deeply branching
dsr
sequences related to environmental
dsr
sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other
dsr
clones were affiliated with gram-positive thermophilic sulfate reducers (genus
Desulfotomaculum
) and the delta-proteobacterial species
Desulforhabdus amnigena
and
Thermodesulforhabdus norvegica
. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with
Desulfobacterium niacini
, a member of the acetate-oxidizing, nutritionally versatile genus
Desulfobacterium
, and one clone related to
Desulfobacula toluolica
and
Desulfotignum balticum
. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and
dsrAB
clone libraries resulted in identification of members of the
Desulfobacteriales
in the Guaymas sediments. In addition, the
dsrAB
sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.
Predictability of
Vibrio cholerae
in Chesapeake Bay
Authors: Louis Valérie R. , Russek-Cohen Estelle , Choopun Nipa , Rivera Irma N. G. , Gangle Brian , Jiang Sunny C. , Rubin Andrea , Patz Jonathan A. , Huq Anwar , Colwell Rita R.
Published: May 2003

Vibrio cholerae
is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish. The correlation between the occurrence of
V. cholerae
in Chesapeake Bay and environmental factors was investigated over a 3-year period. Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000). Enrichment was used to detect culturable
V. cholerae
, and 21.1% (
n
= 427) of the samples were positive. As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics. A direct fluorescent-antibody assay was used to detect
V. cholerae
O1, and 23.8% (
n
= 412) of the samples were positive.
V. cholerae
was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower. Statistical models successfully predicted the presence of
V. cholerae
as a function of water temperature and salinity. Temperatures above 19°C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable
V. cholerae
. The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of
V. cholerae
and that salinity is a useful indicator. Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of
V. cholerae
in Chesapeake Bay.
Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups
Authors: Nadeau Lloyd J. , He Zhongqi , Spain Jim C.
Published: May 2003

Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from
Pseudomonas pseudoalcaligenes
JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from
Ralstonia eutropha
JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from
Comamonas acidovorans
NBA-10 and
Pseudomonas
sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H
2
18
O did not indicate any
18
O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from
R. eutropha
JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from
Pseudomonas
sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was
18
O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in
Pseudomonas
strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.
Origin of Contamination and Genetic Diversity of
Escherichia coli
in Beef Cattle
Authors: Aslam Mueen , Nattress Frances , Greer Gordon , Yost Chris , Gill Colin , McMullen Lynn
Published: May 2003

The possible origin of beef contamination and genetic diversity of
Escherichia coli
populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the
fliC
gene.
E. coli
was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403
E. coli
isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef.
E. coli
genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the
E. coli
population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the
fliC
gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the
E. coli
contaminating meat can originate from cattle feces, and the
E. coli
population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.
The
ars
Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable
Shewanella
Species Strain ANA-3
Authors: Saltikov Chad W. , Cifuentes Ana , Venkateswaran Kasthuri , Newman Dianne K.
Published: May 2003

Arsenate [As(V); HAsO
4
2−
] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus
Shewanella
and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO
2
]. The generation time and lactate molar growth yield (Y
lactate
) are 2.8 h and 10.0 g of cells mol of lactate
−1
, respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an
ars
operon (
arsDABC
) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains
Shewanella oneidensis
MR-1 and
Escherichia coli
AW3110. When the gene encoding the As(III) efflux pump,
arsB
, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of
arsC
, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y
lactate
value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.
Purification and Characterization of an Inverting Stereo- and Enantioselective
sec
-Alkylsulfatase from the Gram-Positive Bacterium
Rhodococcus ruber
DSM 44541
Authors: Pogorevc Mateja , Faber Kurt
Published: May 2003

Whole cells of
Rhodococcus ruber
DSM 44541 were found to hydrolyze (±)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two
sec
-alkylsulfatases and (at least) one
prim
-alkylsulfatase in the absence of an inducer, such as a
sec
-alkyl sulfate or a
sec
-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30°C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (
R
)-2-octyl sulfate furnished (
S
)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear
sec
-alkyl sulfates (C
7
to C
10
) were freely accepted, no activity was found with branched and mixed aryl-alkyl
sec
-sulfates. Due to the fact that
prim
-sulfates were not accepted, the enzyme was classified as
sec-
alkylsulfatase (EC 3.1.6.X).
Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe
Authors: Oehl Fritz , Sieverding Ewald , Ineichen Kurt , Mäder Paul , Boller Thomas , Wiemken Andres
Published: May 2003

The impact of land use intensity on the diversity of arbuscular mycorrhizal fungi (AMF) was investigated at eight sites in the “three-country corner” of France, Germany, and Switzerland. Three sites were low-input, species-rich grasslands. Two sites represented low- to moderate-input farming with a 7-year crop rotation, and three sites represented high-input continuous maize monocropping. Representative soil samples were taken, and the AMF spores present were morphologically identified and counted. The same soil samples also served as inocula for “AMF trap cultures” with
Plantago lanceolata
,
Trifolium pratense
, and
Lolium perenne
. These trap cultures were established in pots in a greenhouse, and AMF root colonization and spore formation were monitored over 8 months. For the field samples, the numbers of AMF spores and species were highest in the grasslands, lower in the low- and moderate-input arable lands, and lowest in the lands with intensive continuous maize monocropping. Some AMF species occurred at all sites (“generalists”); most of them were prevalent in the intensively managed arable lands. Many other species, particularly those forming sporocarps, appeared to be specialists for grasslands. Only a few species were specialized on the arable lands with crop rotation, and only one species was restricted to the high-input maize sites. In the trap culture experiment, the rate of root colonization by AMF was highest with inocula from the permanent grasslands and lowest with those from the high-input monocropping sites. In contrast, AMF spore formation was slowest with the former inocula and fastest with the latter inocula. In conclusion, the increased land use intensity was correlated with a decrease in AMF species richness and with a preferential selection of species that colonized roots slowly but formed spores rapidly.
Association of
Borrelia garinii
and
B. valaisiana
with Songbirds in Slovakia
Authors: Hanincová Klára , Taragelová Veronika , Koci Juraj , Schäfer Stefanie M. , Hails Rosie , Ullmann Amy J. , Piesman Joseph , Labuda Milan , Kurtenbach Klaus
Published: May 2003

In Europe, 6 of the 11 genospecies of
Borrelia burgdorferi
sensu lato are prevalent in questing
Ixodes ricinus
ticks. In most parts of Central Europe,
B. afzelii
,
B. garinii
, and
B. valaisiana
are the most frequent species, whereas
B. burgdorferi
sensu stricto,
B. bissettii
, and
B. lusitaniae
are rare. Previously, it has been shown that
B. afzelii
is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of
B. garinii
and
B. valaisiana
in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing
I. ricinus
ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as
B. garinii
and
B. valaisiana
, whereas questing ticks were infected with
B. afzelii
,
B. garinii
, and
B. valaisiana
. Furthermore, identical
ospA
alleles of
B. garinii
were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of
B. garinii
and
B. valaisiana
but not of
B. afzelii
. This and previous studies confirm that
B. burgdorferi
sensu lato is host associated and that this bacterial species complex contains different ecotypes.
Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in
Clostridium acetobutylicum
ATCC 824
Authors: Zhao Yinsuo , Hindorff Lucia A. , Chuang Amy , Monroe-Augustus Melanie , Lyristis Michael , Harrison Mary L. , Rudolph Frederick B. , Bennett George N.
Published: May 2003

The cyclopropane fatty acid synthase gene (
cfa
) of
Clostridium acetobutylicum
ATCC 824 was cloned and overexpressed under the control of the clostridial
ptb
promoter. The function of the
cfa
gene was confirmed by complementation of an
Escherichia coli cfa
-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing
cfa
were introduced into
C. acetobutylicum
hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the
cfa
gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the
cfa
-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of
cfa
results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a
marR
homologous gene preceding the
cfa
gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.
Recovery and Enumeration of
Cryptosporidium parvum
from Animal Fecal Matrices
Authors: Davies Cheryl M. , Kaucner Christine , Deere Daniel , Ashbolt Nicholas J.
Published: May 2003

Accurate quantification of
Cryptosporidium parvum
oocysts in animal fecal deposits on land is an essential starting point for estimating watershed
C. parvum
loads. Due to the general poor performance and variable recovery efficiency of existing enumeration methods, protocols were devised based on initial dispersion of oocysts from feces by vortexing in 2 mM tetrasodium pyrophosphate, followed by immunomagnetic separation. The protocols were validated by using an internal control seed preparation to determine the levels of oocyst recovery for a range of fecal types. The levels of recovery of 10
2
oocysts from cattle feces (0.5 g of processed feces) ranged from 31 to 46%, and the levels of recovery from sheep feces (0.25 g of processed feces) ranged from 21% to 35%. The within-sample coefficients of variation for the percentages of recovery from five replicates ranged from 10 to 50%. The ranges for levels of recovery of oocysts from cattle, kangaroo, pig, and sheep feces (juveniles and adults) collected in a subsequent watershed animal fecal survey were far wider than the ranges predicted by the validation data. Based on the use of an internal control added to each fecal sample, the levels of recovery ranged from 0 to 83% for cattle, from 4 to 62% for sheep, from 1 to 42% for pigs, and from 40 to 73% for kangaroos. Given the variation in the levels of recovery of oocysts from different fecal matrices, it is recommended that an internal control be added to at least one replicate of every fecal sample analyzed to determine the percentage of recovery. Depending on the animal type and based on the lowest approximate percentages of recovery, between 10 and 100 oocysts g of feces
−1
must be present to be detected.
Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays
Authors: Urakawa Hidetoshi , El Fantroussi Said , Smidt Hauke , Smoot James C. , Tribou Erik H. , Kelly John J. , Noble Peter A. , Stahl David A.
Published: May 2003

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of
Staphylococcus epidermidis
(38 nucleotides) and
Nitrosomonas eutropha
(39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.
A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria
Authors: Holm Claus , Jespersen Lene
Published: May 2003

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with
Staphylococcus aureus
and
Escherichia coli
. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.
In Vivo Tracking ofCampylobacter jejuniby Using a Novel Recombinant Expressing Green Fluorescent Protein
Authors: Mixter Philip F. , Klena John D. , Flom Gary A. , Siegesmund Amy M. , Konkel Michael E.
Published: May 2003

Campylobacter jejuniis a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study ofC. jejuniin a variety of niches.C. jejunitransformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally withC. jejuniharboring thegfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined thatC. jejunisynthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+Gr-1+lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+Gr-1−lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b−CD45R+B lymphocytes from the lavage of theC. jejuni-injected mice were associated with green-fluorescentC. jejuni(proportions ranged from 0.75 to 0.77%). Cell-freeC. jejuniwas recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of theC. jejuniF38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate thatC. jejuniassociates with marker-defined cellular subsets in vivo with a novelgfpreporter system and thatC. jejunigenotypic variants can be isolated from both in vitro and in vivo systems.
Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods
Authors: Thimm Torsten , Tebbe Christoph C.
Published: May 2003

A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of
Folsomia candida
(Collembola) and the detection of bacteria in both the gut and tissue.
Specific Detection of
Dehalococcoides
Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes
Authors: Yang Yanru , Zeyer Josef
Published: May 2003

Dehalococcoides ethenogenes
is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of
Dehalococcoides
species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for
Dehalococcoides
species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except
D. ethenogenes
and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of
Dehalococcoides
species. In situ hybridization of probe Dhe1259t with
D. ethenogenes
strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active
Dehalococcoides
species in these enrichment samples.
RecombinantSaccharomyces cerevisiaeExpressing P450 in Artificial Digestive Systems: a Model for Biodetoxication in the Human Digestive Environment
Authors: Blanquet S. , Meunier J. P. , Minekus M. , Marol-Bonnin S. , Alric M.
Published: May 2003

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model ofSaccharomyces cerevisiaeexpressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% ± 10.1% [n= 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% ± 2.7% [n= 3]). For the first time, the ability of recombinantS. cerevisiaeto carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% ± 5.8% (n= 3) of the ingestedtrans-cinnamic acid was converted intop-coumaric acid after 4 h of digestion, as well as 8.9% ± 1.6% (n= 3) in the stomach, 13.8% ± 3.3% (n= 3) in the duodenum, 11.8% ± 3.4% (n= 3) in the jejunum, and 6.5% ± 1.0% (n= 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest thatS. cerevisiaemay afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.
Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68°C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation
Authors: Ferris Mike J. , Kühl Michael , Wieland Andrea , Ward David M.
Published: May 2003

We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68°C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O2and oxygenic photosynthesis demonstrated the existence of physiologically distinctSynechococcuspopulations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation inSynechococcus16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths.
In Situ Accessibility of
Saccharomyces cerevisiae
26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains
Authors: Inácio João , Behrens Sebastian , Fuchs Bernhard M. , Fonseca Álvaro , Spencer-Martins Isabel , Amann Rudolf
Published: May 2003

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of
Saccharomyces cerevisiae
PYCC 4455
T
. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.
Isolation and Characterization of Novel Psychrophilic, Neutrophilic, Fe-Oxidizing, Chemolithoautotrophic α- and γ-
Proteobacteria
from the Deep Sea
Authors: Edwards K. J. , Rogers D. R. , Wirsen C. O. , McCollom T. M.
Published: May 2003

We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS
2
), basalt glass (∼10 wt% FeO), and siderite (FeCO
3
), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-
Proteobacteria
most closely related to the heterotrophic bacterium
Marinobacter aquaeolei
(87 to 94% sequence similarity). A second group of isolates are α-
Proteobacteria
most closely related to the deep-sea heterotrophic bacterium
Hyphomonas jannaschiana
(81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.
Development of a Plating Medium for Selection of
Helicobacter pylori
from Water Samples
Authors: Degnan A. J. , Sonzogni W. C. , Standridge J. H.
Published: May 2003

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of
Helicobacter pylori
. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (
Acinetobacter
,
Aeromonas
,
Bacillus
,
Escherichia coli
,
Enterobacter
,
Enterococcus
,
Helicobacter pylori
, and
Pseudomonas
strains) were individually plated onto each of these media. Three organisms (
Acinetobacter
,
E. coli
, and
H. pylori
) were able to grow on all five media. This growth was unacceptable since
Helicobacter
grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (
Flavobacterium
,
Serratia
,
Citrobacter
,
Pasteurella
,
Ochrobactrum
,
Rahnella
, and unidentified molds) and were further adulterated with the eight strains listed above (10
6
CFU of each strain per 100 ml) were spiked with
H. pylori
and were plated. In spite of the heavy mixed microbial load, only
H. pylori
colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of
H. pylori
colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for
H. pylori
from mixed microbial populations in water and reduces the time required to complete the assay.
Environmental Surveillance System To Track Wild Poliovirus Transmission
Authors: Deshpande Jagadish M. , Shetty Sushmitha J. , Siddiqui Zaeem A.
Published: May 2003

Eradication of poliomyelitis from large metropolis cities in India has been difficult due to high population density and the presence of large urban slums. Three paralytic poliomyelitis cases were reported in Mumbai, India, in 1999 and 2000 in spite of high immunization coverage and good-quality supplementary immunization activities. We therefore established a systematic environmental surveillance study by weekly screening of sewage samples from three high-risk slum areas to detect the silent transmission of wild poliovirus. In 2001, from among the 137 sewage samples tested, wild poliovirus type 1 was isolated from 35 and wild poliovirus type 3 was isolated from 1. Acute flaccid paralysis (AFP) surveillance indicated one case of paralytic poliomyelitis from the city. Phylogenetic analysis with complete VP1 sequences revealed that the isolates from environmental samples belonged to four lineages of wild polioviruses recently isolated from poliomyelitis cases in Uttar Pradesh and not to those previously isolated from AFP cases in Mumbai. Wild poliovirus thus introduced caused one case of paralytic poliomyelitis. The virus was detected in environmental samples 3 months before. It was found that wild polioviruses introduced several times during the year circulated in Mumbai for a limited period before being eliminated. Environmental surveillance was found to be sensitive for the detection of wild poliovirus silent transmission. Nucleotide sequence analysis helped identify wild poliovirus reservoir areas.