bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Distinct
Campylobacter lari
Genogroups by Amplified Fragment Length Polymorphism and Protein Electrophoretic Profiles
Authors: Duim Birgitta , Wagenaar Jaap A. , Dijkstra Jeroen R. , Goris Johan , Endtz Hubert P. , Vandamme Peter A. R.
Published: January 2004

Campylobacter lari
is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters (NARTC) and the biochemical
C. lari
variants, including the urease-positive campylobacters (UPTC), the nalidixic acid-susceptible campylobacters (NASC), and the urease-producing nalidixic acid-susceptible campylobacters. To study the taxonomic and epidemiological relationships among strains of the
C. lari
variants, amplified fragment length polymorphism (AFLP) profiling and whole-cell protein profile analysis were performed with 55
C. lari
strains. Great genetic heterogeneity in AFLP and protein profiles was observed. Numerical analysis of AFLP profiles and of partial protein profiles allowed discrimination of four distinct genogroups. AFLP cluster I included nearly homogeneous patterns for
C. lari
NARTC strains (genogroup I). UPTC strains together with non-urease-producing NASC strains produced highly diverse patterns and were placed in genogroup II. The genogroup III strains had the NASC phenotype and produced more homogeneous patterns. Finally, genogroup IV strains had the classical NARTC phenotype and produced AFLP patterns that were very distinct from those of other genogroups. One UPTC strain had aberrant patterns and clustered separately, which may indicate that there is an additional genogroup. Preliminary DNA-DNA hybridization experiments suggested that genogroups I and III represent a single genomic species and that genogroup IV represents a distinct species. The detection of moderate levels of DNA-DNA hybridization between a genogroup II reference strain and genogroup I and III reference strains highlights the need for further DNA-DNA hybridization experiments to clarify the taxonomic status of the former group. No correlation of genogroups with different sources of strains was identified. These data show that UPTC strains are genetically diverse and distinct from NARTC strains. In addition, they indicate that the classical NARTC phenotype encompasses at least two genogroups.
Biodiversity among
Lactobacillus helveticus
Strains Isolated from Different Natural Whey Starter Cultures as Revealed by Classification Trees
Authors: Gatti Monica , Trivisano Carlo , Fabrizi Enrico , Neviani Erasmo , Gardini Fausto
Published: January 2004

Lactobacillus helveticus
is a homofermentative thermophilic lactic acid bacterium used extensively for manufacturing Swiss type and aged Italian cheese. In this study, the phenotypic and genotypic diversity of strains isolated from different natural dairy starter cultures used for Grana Padano, Parmigiano Reggiano, and Provolone cheeses was investigated by a classification tree technique. A data set was used that consists of 119
L. helveticus
strains, each of which was studied for its physiological characters, as well as surface protein profiles and hybridization with a species-specific DNA probe. The methodology employed in this work allowed the strains to be grouped into terminal nodes without difficult and subjective interpretation. In particular, good discrimination was obtained between
L. helveticus
strains isolated, respectively, from Grana Padano and from Provolone natural whey starter cultures. The method used in this work allowed identification of the main characteristics that permit discrimination of biotypes. In order to understand what kind of genes could code for phenotypes of technological relevance, evidence that specific DNA sequences are present only in particular biotypes may be of great interest.
Multilocus Sequence Typing Scheme for Bacteria of the
Bacillus cereus
Group
Authors: Helgason Erlendur , Tourasse Nicolas J. , Meisal Roger , Caugant Dominique A. , Kolstø Anne-Brit
Published: January 2004

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the
Bacillus cereus
group. This group, which includes the species
B. cereus, B. thuringiensis, B. weihenstephanensis
,
and B. anthracis
, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes,
adk
,
ccpA
,
ftsA
,
glpT
,
pyrE
,
recF
, and
sucC
, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the
B. cereus
group is weakly clonal. In particular, all five
B. anthracis
isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the
B. cereus
group.
Phylogenetic and Physiological Diversity of Microorganisms Isolated from a Deep Greenland Glacier Ice Core
Authors: Miteva V. I. , Sheridan P. P. , Brenchley J. E.
Published: January 2004

We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives,
Proteobacteria
, and the
Cytophaga-Flavobacterium-Bacteroides
group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.
Within- and between-Lake Variability in the Composition of Bacterioplankton Communities: Investigations Using Multiple Spatial Scales
Authors: Yannarell Anthony C. , Triplett Eric W.
Published: January 2004

This study examined the similarity of epilimnetic bacterial community composition (BCC) across several within- and among-lake spatial scales, and the environmental factors giving rise to similar bacterial communities in different lakes were also explored. Samples were collected from 13 northern and southern Wisconsin lakes representing gradients in lake size, productivity, dissolved organic carbon and humic acid contents, and pH. Hypotheses regarding patchy distribution of bacterial communities in lakes were tested by comparing samples collected from nearby (tens of meters) and distant (hundreds of meters) sampling sites in the same lake. BCC was characterized by using a molecular fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA). Overall, samples collected at the 10-m, 100-m, and between-lake scales differed by 13, 17, and 75%, respectively. Variation at these last two scales was significant. The development of within-lake variation in BCC appeared to depend on the isolation of water by lake shoreline features such as bays or narrow constrictions. ARISA profiles from northern lakes had fewer peaks and were less similar to each other than were those of the southern lakes, suggesting that regional features do not necessarily lead to the development of similar bacterial communities. Lakes at similar positions on productivity and dissolved organic carbon concentration gradients had similar bacterial communities, and bacterial diversity was positively correlated with lake productivity and water temperature. Factorial studies taking into account these gradients, as well as regional spatial scales, should provide much insight into the nature of aquatic bacterial biogeography.
Novel Pathway for Utilization of Cyclopropanecarboxylate by
Rhodococcus rhodochrous
Authors: Toraya Tetsuo , Oka Takayuki , Ando Manabu , Yamanishi Mamoru , Nishihara Hiroshi
Published: January 2004

A new strain isolated from soil utilizes cyclopropanecarboxylate as the sole source of carbon and energy and was identified as
Rhodococcus rhodochrous
(H. Nishihara, Y. Ochi, H. Nakano, M. Ando, and T. Toraya, J. Ferment. Bioeng. 80:400-402, 1995). A novel pathway for the utilization of cyclopropanecarboxylate, a highly strained compound, by this bacterium was investigated. Cyclopropanecarboxylate-dependent reduction of NAD
+
in cell extracts of cyclopropanecarboxylate-grown cells was observed. When intermediates accumulated in vitro in the absence of NAD
+
were trapped as hydroxamic acids by reaction with hydroxylamine, cyclopropanecarboxohydroxamic acid and 3-hydroxybutyrohydroxamic acid were formed. Cyclopropanecarboxyl-coenzyme A (CoA), 3-hydroxybutyryl-CoA, and crotonyl-CoA were oxidized with NAD
+
in cell extracts, whereas methacrylyl-CoA and 3-hydroxyisobutyryl-CoA were not. When both CoA and ATP were added, organic acids corresponding to the former three CoA thioesters were also oxidized in vitro by NAD
+
, while methacrylate, 3-hydroxyisobutyrate, and 2-hydroxybutyrate were not. Therefore, it was concluded that cyclopropanecarboxylate undergoes oxidative degradation through cyclopropanecarboxyl-CoA and 3-hydroxybutyryl-CoA. The enzymes catalyzing formation and ring opening of cyclopropanecarboxyl-CoA were shown to be inducible, while other enzymes involved in the degradation were constitutive.
Comparative Metabolic Flux Analysis of Lysine-Producing
Corynebacterium glutamicum
Cultured on Glucose or Fructose
Authors: Kiefer Patrick , Heinzle Elmar , Zelder Oskar , Wittmann Christoph
Published: January 2004

A comprehensive approach to
13
C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing
Corynebacterium glutamicum
on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS
Fructose
) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTS
Mannose
was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of
C. glutamicum
on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by
C. glutamicum
on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.
New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil
Authors: Bürgmann Helmut , Widmer Franco , Von Sigler William , Zeyer Josef
Published: January 2004

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the
nifH
marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all
nifH
genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same
nifH
fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified
nifH
sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.
Generation and Utilization of Polyclonal Antibodies to a Synthetic C-Terminal Amino Acid Fragment of Divercin V41, a Class IIa Bacteriocin
Authors: Richard Christelle , Drider Djamel , Fliss Ismael , Denery Sandra , Prevost Herve
Published: January 2004

Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized C-terminal part of divercin V41 (DvnCt) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and reactivity of the DvnCt-KLH-generated antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) using supernatant from cultures of 13 representative lactic acid bacterium strains, and specificity was confirmed by Western blot analysis. Anti-DvnCt-KLH antibodies were able to recognize not only divercin V41 but also enterocin P and piscicocin V1b, two other members of the class IIa bacteriocins. Production and activity of DvnV41 were evaluated by ELISA and activity tests during the growth of
Carnobacterium divergens
V41 in MRS medium containing or not containing Tween 80. Divercin V41, enterocin P, and piscicocin V1b were therefore purified by a single-step immunoaffinity chromatography method using a Sepharose matrix CNBr-activated directed binding of anti-DvnCt-KLH polyclonal antibodies.
Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication
Authors: Lam Phyllis , Cowen James P.
Published: January 2004

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.
Genetic Structure and Population Dynamics of
Xanthomonas axonopodis
pv. manihotis in Colombia from 1995 to 1999
Authors: Restrepo Silvia , Velez Claudia M. , Duque Myriam C. , Verdier Valérie
Published: January 2004

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen
Xanthomonas axonopodis
pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different
X. axonopodis
pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the
X. axonopodis
pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of
X. axonopodis
pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of
X. axonopodis
pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.
Characterization of Lipopolysaccharides Present in Settled House Dust
Authors: Park Ju-Hyeong , Szponar Bogumila , Larsson Lennart , Gold Diane R. , Milton Donald K.
Published: January 2004

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a
Limulus
assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C
16:0
and C
18:0
) 3-OHFAs were predominant in HD compared with short-chain (C
10:0
, C
12:0
, and C
14:0
) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C
16:0
was negatively correlated and 3-OH C
18:0
was not correlated with endotoxin activity, consistent with previous findings that the
Limulus
assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C
10:0
, the highest endotoxin activities, and the highest specific activities (
P
< 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (
P
= 0.03) compared with single-family homes in study 2. These data suggest that the
Limulus
assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.
Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates
Authors: Dumont Frédéric , Marechal Pierre-André , Gervais Patrick
Published: January 2004

This work studied the viabilities of five types of cells (two yeast cells,
Saccharomyces cerevisiae
CBS 1171 and
Candida utilis
; two bacterial strains,
Escherichia coli
and
Lactobacillus plantarum
; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000°C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180°C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000°C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000°C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.
WolbachiaTransfer fromRhagoletis cerasitoDrosophila simulans: Investigating the Outcomes of Host-Symbiont Coevolution
Authors: Riegler Markus , Charlat Sylvain , Stauffer Christian , Merçot Hervé
Published: January 2004

Wolbachiais an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bearWolbachiaare mated with females that do not. In the cherry fruit fly,Rhagoletis cerasi,Wolbachiaseems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of twoWolbachiavariants (wCer1 andwCer2) fromR. cerasiintoDrosophila simulans, which was performed with two major goals in mind: first, to isolatewCer1 fromwCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recentWolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring.wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, whilewCer2 was established in the new host. Transmission rates ofwCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neitherwCer1 norwCer2 could be naturally maintained inD. simulans. The experiment thus suggests that naturalWolbachiatransfer between species might be restricted by many factors, should the ecological barriers be bypassed.
Characterization of a New
erm
-Related Macrolide Resistance Gene Present in Probiotic Strains of
Bacillus clausii
Authors: Bozdogan Bülent , Galopin Sébastien , Leclercq Roland
Published: January 2004

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four
Bacillus clausii
strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of
B. clausii
, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called
erm
(34), which is related to the
erm
genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of
B. clausii
DSM8716 into
Escherichia coli
. The deduced amino acid sequence presented 61% identity with that of Erm(D) from
B. licheniformis
,
B. halodurans
, and
B. anthracis
. Pulsed-field gel electrophoresis of total DNA digested by I-
Ceu
I, followed by hybridization with an
erm
(34)-specific probe, indicated a chromosomal location of the gene in all
B. clausii
strains. Repeated attempts to transfer resistance to macrolides by conjugation from
B. clausii
strains to
Enterococcus faecalis
JH2-2,
E. faecium
HM1070, and
B. subtilis
UCN19 were unsuccessful.
Nitrobenzoates and Aminobenzoates Are Chemoattractants for
Pseudomonas
Strains
Authors: Parales Rebecca E.
Published: January 2004

Three
Pseudomonas
strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays.
Pseudomonas putida
PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the β-ketoadipate pathway, has a well-characterized β-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response.
P. putida
TW3 and
Pseudomonas
sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the
ortho
(β-ketoadipate) and
meta
pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the β-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (β-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a β-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.
Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods
Authors: Broderick Nichole A. , Raffa Kenneth F. , Goodman Robert M. , Handelsman Jo
Published: January 2004

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria disparL.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the γ-Proteobacteriaand low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the α-Proteobacterium,Actinobacterium, andCytophaga/Flexibacter/Bacteroidesdivisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that anEnterococcusspecies and culture-independent analysis indicated that anEntbactersp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.
Nitrous Oxide Reductase (
nosZ
) Gene Fragments Differ between Native and Cultivated Michigan Soils
Authors: Stres Blaž , Mahne Ivan , Avguštin Gorazd , Tiedje James M.
Published: January 2004

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (
nosZ
) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions.
nosZ
gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total
nosZ
RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total
nosZ
profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify
nosZ
identity. The sequence diversity of
nosZ
clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct
nosZ
RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for
nosZ
RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of
nosZ
clones into an interleaved Michigan soil cluster whose cultured members are α-
Proteobacteria
. Only four
nosZ
sequences from cultivated soil and one from the native soil were related to sequences found in γ-
Proteobacteria.
Sequences from the native field formed a distinct, closely related cluster (
D
mean
= 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (
D
mean
= 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of
nosZ
sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.
Selection and Characterization of Conditionally Active Promoters in
Lactobacillus plantarum
, Using Alanine Racemase as a Promoter Probe
Authors: Bron Peter A. , Hoffer Sally M. , Van Swam Iris I. , De Vos Willem M. , Kleerebezem Michiel
Published: January 2004

This paper describes the use of the
alr
gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in
Lactobacillus plantarum
. Random fragments of the
L. plantarum
WCFS1 genome were cloned upstream of the promoterless
alr
gene of
Lactococcus lactis
in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an
L. plantarum
Δ
alr
strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the
d
-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 μg of the competitive Alr inhibitor
d
-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of
d
-cycloserine, adding a quantitative factor to the primarily qualitative nature of the
alr
complementation screen. Screening of the
alr
complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.
Use of Antibiotic Susceptibility Patterns and Pulsed-Field Gel Electrophoresis To Compare Historic and Contemporary Isolates of Multi-Drug-Resistant
Salmonella enterica
subsp.
enterica
Serovar Newport
Authors: Berge Anna Catharina B. , Adaska John M. , Sischo William M.
Published: January 2004

Recently, multi-drug-resistant (MDR)
Salmonella enterica
subspecies
enterica
serovar Newport reemerged as a public and animal health problem. The antibiotic resistance of 198 isolates and the pulsed-field gel electrophoresis patterns (PFGE) of 139 isolates were determined. Serovar Newport isolates collected between 1988 and 2001 were included in the study. One hundred seventy-eight isolates were collected from the San Joaquin valley in California and came from dairy cattle clinical samples, human clinical samples, bulk tank milk samples, fecal samples from preweaned calves, and waterways. Twenty clinical isolates from humans from various regions of the United States were also included in the study. Resistance to 18 antibiotics was determined using a disk diffusion assay. PFGE patterns were determined using a single enzyme (
Xba
I). The PFGE and antibiogram patterns were described using cluster analysis. Although the antibiotic resistance patterns of historic (1988 to 1995) and contemporary (1999 to 2001) isolates were similar, the contemporary isolates differed from the historic isolates by being resistant to cephalosporins and florfenicol and in their general sensitivity to kanamycin and neomycin. With few exceptions, the contemporary isolates clustered together and were clearly separated from the historic isolates. One PFGE-antibiogram cluster combination was predominant for the recent isolates, which were taken from human samples from all parts of the United States, as well as in the isolates from California, indicating a rapid dissemination of this phenotypic strain. The data are consistent with the hypothesis that the reemergence of MDR serovar Newport is not simply an acquisition of further antibiotic resistance genes by the historic isolates but reflects a different genetic lineage.
Differential Stress-Induced Regulation of Two Quinone Reductases in the Brown Rot BasidiomyceteGloeophyllum trabeum
Authors: Cohen Roni , Suzuki Melissa R. , Hammel Kenneth E.
Published: January 2004

Quinone reductases (QRDs) have two important functions in the basidiomyceteGloeophyllum trabeum, which causes brown rot of wood. First, a QRD is required to generate biodegradative hydroxyl radicals via redox cycling between twoG. trabeumextracellular metabolites, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ). Second, because 2,5-DMBQ is cytotoxic and 2,5-DMHQ is not, a QRD is needed to maintain the intracellular pool of these metabolites in the reduced form. Given their importance inG. trabeummetabolism, QRDs could prove useful targets for new wood preservatives. We have identified twoG. trabeumgenes, each existing in two closely related, perhaps allelic variants, that encode QRDs in the flavodoxin family. Past work with QRD1 and heterologous expression of QRD2 in this study confirmed that both genes encode NADH-dependent, flavin-containing QRDs. Real-time reverse transcription PCR analyses of liquid- and wood-grown cultures showed thatqrd1expression was maximal during secondary metabolism, coincided with the production of 2,5-DMBQ, and was moderately up-regulated by chemical stressors such as quinones. By contrast,qrd2expression was maximal during fungal growth when 2,5-DMBQ levels were low, yet was markedly up-regulated by chemical stress or heat shock. The total QRD activity in lysates ofG. trabeummycelium was significantly enhanced by induction beforehand with a cytotoxic quinone. The promoter ofqrd2contains likely antioxidant, xenobiotic, and heat shock elements, absent inqrd1, that probably explain the greater response ofqrd2transcription to stress. We conclude from these results that QRD1 is the enzymeG. trabeumroutinely uses to detoxify quinones during incipient wood decay and that it could also drive the biodegradative quinone redox cycle. However, QRD2 assumes a more important role when the mycelium is stressed.
Successful Predation of Filamentous Bacteria by a Nanoflagellate Challenges Current Models of Flagellate Bacterivory
Authors: Wu Qinglong L. , Boenigk Jens , Hahn Martin W.
Published: January 2004

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate,
Ochromonas
sp. strain DS, to test these assumptions. The five strains, including one spirochete and four
Betaproteobacteria
strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 μm. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.
Variable Bacteriocin Production in the Commercial Starter
Lactococcus lactis
DPC4275 Is Linked to the Formation of the Cointegrate Plasmid pMRC02
Authors: Trotter Maeve , McAuliffe Olivia E. , Fitzgerald Gerald F. , Hill Colin , Ross R. Paul , Coffey Aidan
Published: January 2004

Lactococcus lactis
DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with
L. lactis
MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage
c
2 infection of MG1363(pMRC02). While this locus is flanked by IS
946
elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.
Degradation of Benzo[
a
]pyrene by
Mycobacterium vanbaalenii
PYR-1
Authors: Moody Joanna D. , Freeman James P. , Fu Peter P. , Cerniglia Carl E.
Published: January 2004

Metabolism of the environmental pollutant benzo[
a
]pyrene in the bacterium
Mycobacterium vanbaalenii
PYR-1 was examined. This organism initially oxidized benzo[
a
]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[
a
]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were
cis
-4,5-dihydro-4,5-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
cis
-4,5-dihydrodiol),
cis
-11,12-dihydro-11,12-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
cis
-11,12-dihydrodiol),
trans
-11,12-dihydro-11,12-dihydroxybenzo[
a
]pyrene (benzo[
a
]pyrene
trans
-11,12-dihydrodiol), 10-oxabenzo[
def
]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[
a
]pyrene. The
ortho
-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[
a
]pyrene
cis
-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[
a
]pyrene
cis
-4,5-dihydrodiol had 30% 4
S
,5
R
and 70% 4
R
,5
S
absolute stereochemistry. Benzo[
a
]pyrene
cis
-11,12-dihydrodiol adopted an 11
S
,12
R
conformation with 100% optical purity. The enantiomeric composition of benzo[
a
]pyrene
trans
-11,12-dihydrodiol was an equal mixture of 11
S
,12
S
and 11
R
,12
R
molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[
a
]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[
a
]pyrene in
M. vanbaalenii
PYR-1.
Frequency, Size, and Localization of Bacterial Aggregates on Bean Leaf Surfaces
Authors: Monier J.-M. , Lindow S. E.
Published: January 2004

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium
Pseudomonas syringae
pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10
4
cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10
3
cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.
Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic
Escherichia coli
in Agricultural Waste Lagoons
Authors: Chern Eunice C. , Tsai Yu-Li , Olson Betty H.
Published: January 2004

The prevalence among all
Escherichia coli
bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic
E. coli
, and of three genes (
stxI
,
stxII
, and
eaeA
) associated with enterohemorrhagic
E. coli
was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of
E. coli
isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the
stxI
,
stxII
, and
eaeA
genes. The geometric mean prevalence of each toxin gene within the
E. coli
community in waste disposal site waters after nested MPN-PCR was 1:8.5
E. coli
isolates (1:8.5
E. coli
) for the LTIIa toxin gene and 1:4
E. coli
for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes
stxI
,
stxII
, and
eaeA
, was 1:182
E. coli
. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.