bluuebunny/crossref_metadata_2025_split huggingface dataset:
Directed Evolution of Pyruvate Decarboxylase-Negative
Saccharomyces cerevisiae
, Yielding a C
2
-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast
Authors: van Maris Antonius J. A. , Geertman Jan-Maarten A. , Vermeulen Alexander , Groothuizen Matthijs K. , Winkler Aaron A. , Piper Matthew D. W. , van Dijken Johannes P. , Pronk Jack T. 
Published: January 2004

ABSTRACT

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc
−
) strains of
Saccharomyces cerevisiae
an interesting platform for further metabolic engineering of central metabolism. However, Pdc
−
S. cerevisiae
strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C
2
compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc
−
strain without these growth defects. An acetate-independent Pdc
−
mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C
2
independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc
−
S. cerevisiae
mutant (TAM) that could grow in batch cultures (μ
max
= 0.20 h
−1
) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter
−1
was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass
−1
h
−1
during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose
−1
.
Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria
Authors: Matsuki Takahiro , Watanabe Koichi , Fujimoto Junji , Kado Yukiko , Takada Toshihiko , Matsumoto Kazumasa , Tanaka Ryuichiro 
Published: January 2004

ABSTRACTA highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR withBifidobacteriumgenus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106to 10 cells per PCR assay. It was also found that the method was applicable to the detection ofBifidobacteriumin feces when it was present at concentrations of >106cells per g of feces. Concerning the distribution ofBifidobacteriumspecies in intestinal flora, theBifidobacterium adolescentisgroup, theBifidobacterium catenulatumgroup, andBifidobacterium longumwere found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition ofBifidobacteriumspecies in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.
Metabolism of 2,2′- and 3,3′-Dihydroxybiphenyl by the Biphenyl Catabolic Pathway of
Comamonas testosteroni
B-356
Authors: Sondossi M. , Barriault D. , Sylvestre M. 
Published: January 2004

ABSTRACT

The purpose of this investigation was to examine the capacity of the biphenyl catabolic enzymes of
Comamonas testosteroni
B-356 to metabolize dihydroxybiphenyls symmetrically substituted on both rings. Data show that 3,3′-dihydroxybiphenyl is by far the preferred substrate for strain B-356. However, the dihydrodiol metabolite is very unstable and readily tautomerizes to a dead-end metabolite or is dehydroxylated by elimination of water. The tautomerization route is the most prominent. Thus, a very small fraction of the substrate is converted to other hydroxylated and acidic metabolites. Although 2,2′-dihydroxybiphenyl is a poor substrate for strain B-356 biphenyl dioxygenase, metabolites were produced by the biphenyl catabolic enzymes, leading to production of 2-hydroxybenzoic acid. Data show that the major route of metabolism involves, as a first step, a direct dehydroxylation of one of the
ortho
-substituted carbons to yield 2,3,2′-trihydroxybiphenyl. However, other metabolites resulting from hydroxylation of carbons 5 and 6 of 2,2′-dihydroxybiphenyl were also produced, leading to dead-end metabolites.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThis 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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.