bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fluidity Method for the Determination of Association. I
Authors: Bingham Eugene C. , Spooner Laurence W.
Published: January 2006

For many years scientists have tried to correlate the viscosity of pure liquids with their other physical and chemical properties. A relation between viscosity and chemical composition was observed first by Pribřam and Handl. Thorpe and Rodger attempted to assign a value to each atom in 1893. It was shown by Bingham and Harrison that fluidities rather than viscosities are additive, and they devised atomic temperature constants of fluidity for the single fluidity of 200 rhes. The effect of association on fluidity had not been noted heretofore, but Thorpe and Rodger assigned values to the iso-grouping, the double bond linkage, the ring grouping, the hydroxyl oxygen, etc., which denoted the supposed effects of “constitution.” These constitutive effects were at least partly due to association. Bingham and Harrison gave a method for calculating the association and compared the values obtained with the values obtained in other ways. They found that the values to be assigned to a double bond, a ring grouping or an iso-grouping are very small. Bingham and Darrall in a study of the isomeric octyl alcohols went so far as to assume that the constitution of chemical compounds does not affect the fluidity except in so far as it affects the association. It is a very simplifying assumption that the slight difference between the temperature required to give an unsaturated compound a certain fluidity and that required to give the corresponding saturated compound the same fluidity is due to association. The absence of two hydrogen atoms is made up for by the “condition of unsaturation.” Bingham and Fornwalt substantiated the conclusions of Bingham and Darrall and they were able to formulate the trend of association with changes in chemical composition and constitution for a number of types of compounds.
Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Authors: Albers S.-V. , Jonuscheit M. , Dinkelaker S. , Urich T. , Kletzin A. , Tampé R. , Driessen A. J. M. , Schleper C.
Published: January 2006

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote
Sulfolobus solfataricus
, a model organism of hyperthermophilic archaea that grows optimally at 80°C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol.
48:
1241-1252, 2003). Here we have refined this system for high-level gene expression in
S. solfataricus
with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from
Acidianus ambivalens
, an Fe-S protein of the ABC class from
S. solfataricus
, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.
Biotransformation of 1,2,3-Tri- and 1,2,3,4,7,8-Hexachlorodibenzo-p- Dioxin bySphingomonas wittichiiStrain RW1
Authors: Nam In-Hyun , Kim Young-Mo , Schmidt Stefan , Chang Yoon-Seok
Published: January 2006

Sphingomonas wittichiiRW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and aftern-butylboronate andN,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.
Transposons Inactivate Biosynthesis of the Nonribosomal Peptide Microcystin in Naturally Occurring
Planktothrix
spp
Authors: Christiansen Guntram , Kurmayer Rainer , Liu Qian , Börner Thomas
Published: January 2006

The filamentous cyanobacteria
Planktothrix
spp. occur in the temperate region of the Northern hemisphere. The red-pigmented
Planktothrix rubescens
bacteria occur in deep, physically stratified, and less eutrophic lakes.
Planktothrix
is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (
mcy
genes).
Planktothrix
spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of
Planktothrix
strains containing all
mcy
genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the
mcy
genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in
mcyB
; one strain) and 1,869 bp (in
mcyHA
; three strains), and three insertions (IS), spanning 1,429 bp (in
mcyD
; eight strains), 1,433 bp (in
mcyEG
; one strain), and 1,433 bp (in
mcyA
; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in
mcyD
and
mcyA
) and one deletion (in
mcyHA
) were found regularly in populations of
P. rubescens
in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the
mcy
gene pool in
Planktothrix
populations over several years.
Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions
Authors: Koutsoumanis K. , Stamatiou A. , Skandamis P. , Nychas G.-J. E.
Published: January 2006

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by
Brochothrix thermosphacta
, while the other members of the microbial association (e.g., lactic acid bacteria and
Enterobacteriaceae
) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ
max
] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ
max
and λ(μ
max
× λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and
B. thermosphacta
. For the latter bacteria, a negative linear correlation between ln(μ
max
× λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.
Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples
Authors: Grant Susan , Grant William D. , Cowan Don A. , Jones Brian E. , Ma Yanhe , Ventosa Antonio , Heaphy Shaun
Published: January 2006

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at −20°C. Protocols for library construction were established on total RNA extracted from
Acanthamoeba polyphaga
trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.
Slugs: Potential Novel Vectors of
Escherichia coli
O157
Authors: Sproston Emma L. , Macrae M. , Ogden Iain D. , Wilson Michael J. , Strachan Norval J. C.
Published: January 2006

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g.,
Escherichia coli
O157) transfer from animal feces to salad vegetables.
Escherichia coli
O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (
vt1
and
vt2
) and the attaching and effacing gene (
eae
), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an
E. coli
mutant resistant to nalidixic acid showed that the ubiquitous slug species
Deroceras reticulatum
could carry viable
E. coli
on its external surface for up to 14 days. Slugs that had been fed
E. coli
shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that
E. coli
persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of
E. coli
O157.
Occurrence and Genetic Diversity of Uncultured
Legionella
spp. in Drinking Water Treated at Temperatures below 15°C
Authors: Wullings Bart A. , van der Kooij Dick
Published: January 2006

Representatives of the genus
Legionella
were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies.
Legionella
concentrations ranged from 1.1 × 10
3
to 7.8 × 10
5
cells liter
−1
and were significantly higher in SW treated with multiple barriers at 4°C than in GW treated at 9 to 12°C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter
−1
) were detected in treated water by the culture method.
Legionella
was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the
Legionella
concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with
Legionella
sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described
Legionella
species and types, including
L. bozemanii
,
L. worsleiensis
,
Legionella
-like amoebal pathogen types,
L. quateirensis
,
L. waltersii
, and
L. pneumophila
. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of
Legionella
. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15°C.
Deoxysugar Transfer during Chromomycin A
3
Biosynthesis in
Streptomyces griseus
subsp.
griseus
: New Derivatives with Antitumor Activity
Authors: Menéndez Nuria , Nur-e-Alam Mohammad , Fischer Carsten , Braña Alfredo F. , Salas José A. , Rohr Jürgen , Méndez Carmen
Published: January 2006

Chromomycin A
3
is an antitumor drug produced by
Streptomyces griseus
subsp.
griseus
. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-
O
-acetyl-
d
-oliose (sugar A) and 4-
O
-methyl-
d
-oliose (sugar B), and a trisaccharide of
d
-olivose (sugar C),
d
-olivose (sugar D), and 4-
O
-acetyl-
l
-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (
cmmGI
,
cmmGII
,
cmmGIII
, and
cmmGIV
), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-
O
-deacetyl-3A-
O
-acetyl-prechromomycin A4, and 3A-
O
-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two
d
-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A
3
biosynthesis.
Effect of Inhibition of Acetoclastic Methanogenesis on Growth of Archaeal Populations in an Anoxic Model Environment
Authors: Penning Holger , Conrad Ralf
Published: January 2006

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H
2
/CO
2
to total CH
4
production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH
3
F, measured the activity and Gibbs free energy (Δ
G
) of CH
4
production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH
3
F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of
Methanosarcina
spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH
3
F seem to be a suitable method for quantifying acetoclastic CH
4
production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.
Genetic Variability and Distribution of Mating Type Alleles in Field Populations of
Leptosphaeria maculans
from France
Authors: Gout Lilian , Eckert Maria , Rouxel Thierry , Balesdent Marie-Hélène
Published: January 2006

Leptosphaeria maculans
is the most ubiquitous fungal pathogen of
Brassica
crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of
L. maculans
in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m
2
field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All
L. maculans
field populations had a high level of gene diversity (
H
= 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m
2
field plots. Population differentiation among the four field populations was low (

G
ST

< 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of
L. maculans
suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.
New Insights into the Lifestyle of the Cold-Loving SM1 Euryarchaeon: Natural Growth as a Monospecies Biofilm in the Subsurface
Authors: Henneberger Ruth , Moissl Christine , Amann Thomas , Rudolph Christian , Huber Robert
Published: January 2006

In the surface waters of sulfidic springs near Regensburg, Bavaria, Germany, the SM1 euryarchaeon, together with filamentous bacteria, forms the recently described unique string-of-pearls community. In addition to naturally occurring string-of-pearls communities, the growth of these communities was also observed on polyethylene nets provided as an artificial attachment material in the streamlets of springs. In order to learn more about the distribution and origin of the SM1 euryarchaeon and its possible occurrence in the subsurface, polyethylene nets were incubated as deeply as possible in different spring holes. After a short residence time, slime-like, milky drops, almost completely composed of SM1 euryarchaeon, were attached to the nets, indicating that this organism grows independent of a partner in deeper earth layers. A newly designed in situ biofilm trapping system allowed the quantitative harvesting of organisms exhibiting this newly discovered lifestyle of the SM1 euryarchaeon for detailed biological studies. The discovery of naturally occurring archaeal biofilms extends our knowledge of the biology and ecological significance of archaea in their environments.
Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between
Entodinium
Populations in Sheep Offered a Hay-Based Diet
Authors: Skillman Lucy C. , Toovey Andrew F. , Williams Andrew J. , Wright André-Denis G.
Published: January 2006

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (
Entodinium
and
Dasytricha
spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (ε) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of
Entodinium
, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (
R
2
= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed.
Entodinium
represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10
0
and 10
6
entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (
P
= 0.05) 20% change in
Entodinium
populations, 52 sheep per treatment group would be required.
Evaluation of Affymetrix Severe Acute Respiratory Syndrome Resequencing GeneChips in Characterization of the Genomes of Two Strains of Coronavirus Infecting Humans
Authors: Sulaiman Irshad M. , Liu Xin , Frace Michael , Sulaiman Nikhat , Olsen-Rasmussen Melissa , Neuhaus Elizabeth , Rota Paul A. , Wohlhueter Robert M.
Published: January 2006

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.
Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments
Authors: Kimura Hiroyuki , Sugihara Maki , Kato Kenji , Hanada Satoshi
Published: January 2006

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.
Structure and Function of Bacterial Communities Emerging from Different Sources under Identical Conditions
Authors: Langenheder Silke , Lindström Eva S. , Tranvik Lars J.
Published: January 2006

The aim of this study was to compare two major hypotheses concerning the formation of bacterial community composition (BCC) at the local scale, i.e., whether BCC is determined by the prevailing local environmental conditions or by “metacommunity processes.” A batch culture experiment where bacteria from eight distinctly different aquatic habitats were regrown under identical conditions was performed to test to what extent similar communities develop under similar selective pressure. Differently composed communities emerged from different inoculum communities, as determined by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. There was no indication that similarity increased between communities upon growth under identical conditions compared to that for growth at the ambient sampling sites. This suggests that the history and distribution of taxa within the source communities were stronger regulating factors of BCC than the environmental conditions. Moreover, differently composed communities were different with regard to specific functions, such as enzyme activities, but maintained similar broad-scale functions, such as biomass production and respiration.
Effect of Simultaneous Inoculation with Yeast and Bacteria on Fermentation Kinetics and Key Wine Parameters of Cool-Climate Chardonnay
Authors: Jussier Delphine , Dubé Morneau Amélie , Mira de Orduña Ramón
Published: January 2006

Inoculating grape musts with wine yeast and lactic acid bacteria (LAB) concurrently in order to induce simultaneous alcoholic fermentation (AF) and malolactic fermentation (MLF) can be an efficient alternative to overcome potential inhibition of LAB in wines because of high ethanol concentrations and reduced nutrient content. In this study, the simultaneous inoculation of yeast and LAB into must was compared with a traditional vinification protocol, where MLF was induced after completion of AF. For this, two suitable commercial yeast-bacterium combinations were tested in cool-climate Chardonnay must. The time courses of glucose and fructose, acetaldehyde, several organic acids, and nitrogenous compounds were measured along with the final values of other key wine parameters. Sensory evaluation was done after 12 months of storage. The current study could not confirm a negative impact of simultaneous AF/MLF on fermentation success and kinetics or on final wine parameters. While acetic acid concentrations were slightly increased in wines after simultaneous AF/MLF, the differences were of neither practical nor legal significance. No statistically significant differences were found with regard to the final values of pH or total acidity and the concentrations of ethanol, acetaldehyde, glycerol, citric and lactic acids, and the nitrogen compounds arginine, ammonia, urea, citrulline, and ornithine. Sensory evaluation by a semiexpert panel confirmed the similarity of the wines. However, simultaneous inoculation led to considerable reductions in overall fermentation durations. Furthermore, differences of physiological and microbiological relevance were found. Specifically, we report the vinification of “super-dry” wines devoid of glucose and fructose after simultaneous inoculation of yeast and bacteria.
Fourier Transform Infrared and Raman Spectroscopy for Characterization of
Listeria monocytogenes
Strains
Authors: Oust Astrid , Møretrø Trond , Naterstad Kristine , Sockalingum Ganesh D. , Adt Isabelle , Manfait Michel , Kohler Achim
Published: January 2006

The purpose of this study was to characterize the variation in biochemical composition of 89 strains of
Listeria monocytogenes
with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the
L. monocytogenes
strains is discussed.
Effects of
Lactococcus lactis
on Composition of Intestinal Microbiota: Role of Nisin
Authors: Bernbom Nete , Licht Tine Rask , Brogren Carl-Henrik , Jelle Birthe , Johansen Anette H. , Badiola Iker , Vogensen Finn K. , Nørrung Birgit
Published: January 2006

This study examined the ability of (i) pure nisin, (ii) nisin-producing
Lactococcus lactis
strain CHCC5826, and (iii) the non-nisin-producing
L. lactis
strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing
L. lactis
strains significantly increased the number of
Bifidobacterium
cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two
L. lactis
strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.
A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by
halA
from the Cyanobacterium
Arthrospira platensis
Is Closely Related to Its Counterparts from Yeasts and Plants
Authors: Zhang Ju-Yuan , Zou Jie , Bao Qiyu , Chen Wen-Li , Wang Li , Yang Huanming , Zhang Cheng-Cai
Published: January 2006

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene,
halA
, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium
Arthrospira
(
Spirulina
)
platensis
. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from
A. platensis
was overproduced in
Escherichia coli
and used for the characterization of its biochemical properties. HalA was dependent on Mg
2+
for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li
+
(50% inhibitory concentration [IC
50
] = 3.6 mM) but only slightly sensitive to Na
+
(IC
50
= 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li
+
and Na
+
, but was comparable to the PAPase AtAHL (Hal2p-like protein) from
Arabidopsis thaliana
. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of
halA
improved the Li
+
tolerance of
E. coli
, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.
Long Serial Analysis of Gene Expression for Gene Discovery and Transcriptome Profiling in the Widespread Marine Coccolithophore
Emiliania huxleyi
Authors: Dyhrman Sonya T. , Haley Sheean T. , Birkeland Shanda R. , Wurch Louie L. , Cipriano Michael J. , McArthur Andrew G.
Published: January 2006

The abundant and widespread coccolithophore
Emiliania huxleyi
plays an important role in mediating CO
2
exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify
E. huxleyi
genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags.
E. huxleyi
appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of
E. huxleyi
N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.
Modeling and Predicting the Simultaneous Growth ofEscherichia coliO157:H7 and Ground Beef Background Microflora for Various Enrichment Protocols
Authors: Vimont A. , Vernozy-Rozand C. , Montet M. P. , Lazizzera C. , Bavai C. , Delignette-Muller M.-L.
Published: January 2006

The simultaneous growth ofEscherichia coliO157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth andE. colibroth), the presence of novobiocin in the broth, and the incubation temperature (37°C or 40°C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40°C) did not inhibit BM growth, and incubation at 40°C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.
Display of α-Amylase on the Surface of
Lactobacillus casei
Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch
Authors: Narita Junya , Okano Kenji , Kitao Tomoe , Ishida Saori , Sewaki Tomomitsu , Sung Moon-Hee , Fukuda Hideki , Kondo Akihiko
Published: January 2006

We developed a new cell surface engineering system based on the PgsA anchor protein from
Bacillus subtilis
. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of
Lactobacillus casei
by genetically displaying α-amylase from the
Streptococcus bovis
strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying
L. casei
cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant
L. casei
cells with cell surface display of AmyA.
Role of Autochthonous Filamentous Fungi in Bioremediation of a Soil Historically Contaminated with Aromatic Hydrocarbons
Authors: D'Annibale A. , Rosetto F. , Leonardi V. , Federici F. , Petruccioli M.
Published: January 2006

Nine fungal strains isolated from an aged and heavily contaminated soil were identified and screened to assess their degradative potential. Among them,
Allescheriella
sp. strain DABAC 1,
Stachybotrys
sp. strain DABAC 3, and
Phlebia
sp. strain DABAC 9 were selected for remediation trials on the basis of Poly R-478 decolorization associated with lignin-modifying enzyme (LME) production. These autochthonous fungi were tested for the abilities to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal colonization was clearly visible and was confirmed by ergosterol determination. In spite of subalkaline pH conditions and the presence of heavy metals, the autochthonous fungi produced laccase and Mn and lignin peroxidases. No LME activities were detected in control microcosms. All of the isolates led to a marked removal of naphthalene, dichloroaniline isomers,
o
-hydroxybiphenyl, and 1,1′-binaphthalene.
Stachybotrys
sp. strain DABAC 3 was the most effective isolate due to its ability to partially deplete the predominant contaminants 9,10-anthracenedione and 7H-benz[DE]anthracen-7-one. A release of chloride ions was observed in soil treated with either
Allescheriella
sp. strain DABAC 1 or
Stachybotrys
sp. strain DABAC 3, suggesting the occurrence of oxidative dehalogenation. The autochthonous fungi led to a significant decrease in soil toxicity, as assessed by both the
Lepidium sativum
L. germination test and the Collembola mortality test.
Molecular Evidence for Association of
Chlamydiales
Bacteria with Epitheliocystis in Leafy Seadragon (
Phycodurus eques
), Silver Perch (
Bidyanus bidyanus
), and Barramundi (
Lates calcarifer
)
Authors: Meijer Adam , Roholl Paul J. M. , Ossewaarde Jacobus M. , Jones Brian , Nowak Barbara F.
Published: January 2006

Epitheliocystis in leafy seadragon (
Phycodurus eques
), silver perch (
Bidyanus bidyanus
), and barramundi (
Lates calcarifer
), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order
Chlamydiales
in a lineage separate from the family
Chlamydiaceae
. By using in situ RNA hybridization, 16S rRNA
Chlamydiales
-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order
Chlamydiales
by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.
Functional Analysis of Unique Class II Insertion Sequence IS
1071
Authors: Sota Masahiro , Yano Hirokazu , Nagata Yuji , Ohtsubo Yoshiyuki , Genka Hiroyuki , Anbutsu Hisashi , Kawasaki Haruhiko , Tsuda Masataka
Published: January 2006

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS
1071
. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS
1071
has remained unclear. Our study revealed that IS
1071
was only able to transpose at high frequencies in two environmental β-proteobacterial strains,
Comamonas testosteroni
and
Delftia acidovorans
, and not in any of the bacteria examined which belong to the α- and γ-proteobacteria. IS
1071
was found to have the functional features of the class II transposons in that (i) the final product of the IS
1071
transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS
1071
, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a
tnpA
mutation of IS
1071
was efficiently complemented by supplying the wild-type
tnpA
gene in
trans
. Deletion analysis of the IS
1071
IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS
1071
might be different from that of the other well-characterized class II transposons.
In Vitro Alginate Polymerization and the Functional Role of Alg8 in Alginate Production by
Pseudomonas aeruginosa
Authors: Remminghorst Uwe , Rehm Bernd H. A.
Published: January 2006

An enzymatic in vitro alginate polymerization assay was developed by using
14
C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing
Pseudomonas aeruginosa
FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic
alg8
knockout mutants of alginate-overproducing
P. aeruginosa
FRD1 and
P. aeruginosa
PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the
alg8
gene. Surprisingly, this resulted in significant alginate overproduction of the complemented
P. aeruginosa
Δalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis.
1
H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from
Bacillus subtilis
was developed.