bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biogeography and Degree of Endemicity of Fluorescent
Pseudomonas
Strains in Soil
Authors: Cho Jae-Chang , Tiedje James M. 
Published: July 2002

ABSTRACT

Fluorescent
Pseudomonas
strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as
Pseudomonas
sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent
Pseudomonas
genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent
Pseudomonas
genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.
Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of
Erwinia amylovora
in Asymptomatic Plant Material
Authors: Llop Pablo , Bonaterra Anna , Peñalver Javier , López María M. 
Published: July 2002

ABSTRACT

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of
Erwinia amylovora
in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of
E. amylovora
, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in
E. amylovora
hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of
E. amylovora
in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.
Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization
Authors: Sghir Abdelghani , Gramet Genevieve , Suau Antonia , Rochet Violaine , Pochart Philippe , Dore Joel 
Published: July 2002

ABSTRACT

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The
Bacteroides
group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%.
Clostridium leptum
subgroup and
Clostridium coccoides
group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while
Bifidobacterium
and
Lactobacillus
groups made up less than 2%.
Osmoprotection by Pipecolic Acid in
Sinorhizobium meliloti
: Specific Effects of
d
and
l
Isomers
Authors: Gouffi Kamila , Bernard Théophile , Blanco Carlos 
Published: July 2002

ABSTRACT

dl
-Pipecolic acid (
dl
-PIP) promotes growth restoration of
Sinorhizobium meliloti
cells facing inhibitory hyperosmolarity. Surprisingly,
d
and
l
isomers of this imino acid supplied separately were not effective. The uptake of
l
-PIP was significantly favored in the presence of the
d
isomer and by a hyperosmotic stress. Chromatographic analysis of the intracellular solutes showed that stressed cells did not accumulate radiolabeled
l
-PIP. Rather, it participates in the synthesis of the main endogenous osmolytes (glutamate and the dipeptide
N
-acetylglutaminylglutamine amide) during the lag phase, thus providing a means for the stressed cells to recover the osmotic balance.
13
C nuclear magnetic resonance analysis was used to determine the fate of
d
-PIP taken into the cells. In the absence of
l
-PIP, the imported
d
isomer was readily degraded. Supplied together with its
l
isomer,
d
-PIP was accumulated temporarily and thus might contribute together with the endogenous osmolytes to enhance the internal osmotic strength. Furthermore, it started to disappear from the cytosol when the
l
isomer was no longer available in the culture medium (during the late exponential phase of growth). Together, these results show an uncommon mechanism of protection of osmotically stressed cells of
S. meliloti
. It was proved, for the first time, that the presence of the two isomers of the same molecule is necessary for it to manifest an osmoprotective activity. Indeed,
d
-PIP seems to play a major role in cellular osmoadaptation through both its own accumulation and improvement of the utilization of the
l
isomer as an immediate precursor of endogenous osmolytes.
Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing
Lactobacillus rhamnosus
DR20
Authors: Tannock G. W. , Munro K. , Harmsen H. J. M. , Welling G. W. , Smart J. , Gopal P. K. 
Published: July 2002

ABSTRACT

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing
Lactobacillus rhamnosus
DR20 (daily dose, 1.6 × 10
9
lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the
Lactobacillus
population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10
2
cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the
Lactobacillus
and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.
Analysis of Ammonia-Oxidizing Bacteria from Hypersaline Mono Lake, California, on the Basis of 16S rRNA Sequences
Authors: Ward B. B. , Martino D. P. , Diaz M. C. , Joye S. B. 
Published: July 2002

ABSTRACT

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the β subdivision of the division
Proteobacteria
(β-subdivision
Proteobacteria
) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different β-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured
Nitrosomonas
strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the
Nitrosomonas europaea
group that are acclimated to alkaline, high-salinity environments. Primers specific for
Nitrosococcus oceanus
, a marine ammonia-oxidizing bacterium in the γ subdivision of the
Proteobacteria
, did not amplify target from any samples.
Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with
Brachyspira hyodysenteriae
Authors: Leser Thomas D. , Lindecrona Rikke Hvid , Jensen Tim K. , Jensen Bent B. , Møller Kristian 
Published: July 2002

ABSTRACT

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with
Brachyspira hyodysenteriae
, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.
Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria
Authors: Lloyd J. R. , Sole V. A. , Van Praagh C. V. G. , Lovley D. R. 
Published: July 2002

ABSTRACT

The dissimilatory Fe(III)-reducing bacterium
Geobacter sulfurreducens
reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO
2
at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO
2
and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single
Geobacter
sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.
Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site
Authors: Bond Philip L. , Smriga Steven P. , Banfield Jillian F. 
Published: July 2002

ABSTRACT

An unusually thick (∼1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to
Leptospirillum
species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the
Archaea
of the
Thermoplasmales
lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with
Acidimicrobium
were detected. Some of these were closely related to “
Ferromicrobium acidophilus
,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the
Proteobacteria
were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.
Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose
Authors: Fernandez Ana S. , Hashsham Syed A. , Dollhopf Sherry L. , Raskin Lutgarde , Glagoleva Olga , Dazzo Frank B. , Hickey Robert F. , Criddle Craig S. , Tiedje James M. 
Published: July 2002

ABSTRACT

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of
Methanosarcina
-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of
Streptococcus
-related and deeply branching
Spirochaetales
-related SSU rDNA genes, and a high percentage of
Methanosaeta
-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by
Eubacterium
-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with
Streptococcus
-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).
Rapid, Quantitative PCR Monitoring of Growth of
Clostridium botulinum
Type E in Modified-Atmosphere-Packaged Fish
Authors: Kimura B. , Kawasaki S. , Nakano H. , Fujii T. 
Published: July 2002

ABSTRACT

A rapid, quantitative PCR assay (TaqMan assay) which quantifies
Clostridium botulinum
type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the
C. botulinum
type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of
C. botulinum
type E and 18 genera of 42 non-
C. botulinum
type E strains, including strains of
C. botulinum
types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of
C. botulinum
DNA could be monitored (the quantifiable range was 10
2
to 10
8
CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of
C. botulinum
cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.
Development and Characterization of a Xylose-Dependent System for Expression of Cloned Genes in
Bacillus subtilis
: Conditional Complementation of a Teichoic Acid Mutant
Authors: Bhavsar Amit P. , Zhao Xumei , Brown Eric D. 
Published: July 2002

ABSTRACT

We have developed a xylose-dependent expression system for tight and modulated expression of cloned genes in
Bacillus subtilis
. The expression system is contained on plasmid pSWEET for integration at the
amyE
locus of
B. subtilis
and incorporates components of the well-characterized, divergently transcribed xylose utilization operon. The system contains the xylose repressor encoded by
xylR
, the promoter and 5′ portion of
xylA
containing an optimized catabolite-responsive element, and intergenic
xyl
operator sequences. We have rigorously compared this expression system to the isopropyl-β-
d
-thiogalactopyranoside-induced
spac
system using a thermostable β-galactosidase reporter (BgaB) and found the
xyl
promoter-operator to have a greater capacity for modulated expression, a higher induction/repression ratio (279-fold for the
xyl
system versus 24-fold with the
spac
promoter), and lower levels of expression in the absence of an inducer. We have used this system to probe an essential function in wall teichoic acid biosynthesis in
B. subtilis
. Expression of the teichoic acid biosynthesis gene
tagD
, encoding glycerol-3-phosphate cytidylyltransferase, from the xylose-based expression system integrated at
amyE
exhibited xylose-dependent complementation of the temperature-sensitive mutant
tag-12
when grown at the nonpermissive temperature. Plasmid pSWEET thus provides a robust new expression system for conditional complementation in
B. subtilis
.
Aspects of Bioavailability of Mercury for Methylation in Pure Cultures of
Desulfobulbus propionicus
(1pr3)
Authors: Benoit J. M. , Gilmour C. C. , Mason R. P. 
Published: July 2002

ABSTRACT

We have previously hypothesized that sulfide inhibits Hg methylation by decreasing its bioavailability to sulfate-reducing bacteria (SRB), the important methylators of Hg in natural sediments. With a view to designing a bioassay to test this hypothesis, we investigated a number of aspects of Hg methylation by the SRB
Desulfobulbus propionicus
, including (i) the relationship between cell density and methylmercury (MeHg) production, (ii) the time course of Hg methylation relative to growth stage, (iii) changes in the bioavailability of an added inorganic Hg (Hg
I
) spike over time, and (iv) the dependence of methylation on the concentration of dissolved Hg
I
present in the culture. We then tested the effect of sulfide on MeHg production by this microorganism. These experiments demonstrated that under conditions of equal bioavailability, per-cell MeHg production was constant through log-phase culture growth. However, the methylation rate of a new Hg spike dramatically decreased after the first 5 h. This result was seen whether methylation rate was expressed as a fraction of the total added Hg or the filtered Hg
I
concentration, which suggests that Hg bioavailability decreased through both changes in Hg complexation and formation of solid phases. At low sulfide concentration, MeHg production was linearly related to the concentration of filtered Hg
I
. The methylation of filtered Hg
I
decreased about fourfold as sulfide concentration was increased from 10
−6
to 10
−3
M. This decline is consistent with a decrease in the bioavailability of Hg
I
, possibly due to a decline in the dissolved neutral complex, HgS
0
.