bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils
Authors: Eriksson Mikael , Sodersten Erik , Yu Zhongtang , Dalhammar Gunnel , Mohn William W.
Published: January 2003

Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites. Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 μg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils. Under aerobic conditions, lowtemperature severely limited PAH biodegradation. After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs. The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil. Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded. The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil. In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal. Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera
Acidovorax
,
Bordetella
,
Pseudomonas
,
Sphingomonas
, and
Variovorax
. Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies). The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum.
Elevated Abundance of Bacteriophage Infecting Bacteria in Soil
Authors: Ashelford Kevin E. , Day Martin J. , Fry John C.
Published: January 2003

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 107g−1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of aSerratiaphage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 108g−1, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown onSerratiaandPseudomonasselective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.
Multicenter Validation of the Analytical Accuracy ofSalmonellaPCR: towards an International Standard
Authors: Malorny Burkhard , Hoorfar Jeffrey , Bunge Cornelia , Helmuth Reiner
Published: January 2003

As part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific forSalmonellaspecies were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43Salmonellaspp. and 47 non-Salmonellastrains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets theinvAgene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for theinvAprimer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with theinvAtarget gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 104CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded (“blind”) DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific forSalmonellaspp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.
Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F
Authors: Schmidt James J. , Stafford Robert G.
Published: January 2003

The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P
1
and P
3
′ residues were substituted with 2,4-dinitrophenyl-lysine and
S
-(
N
-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a

K
i

value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.
Plasmid-Encoded Diacetyl (Acetoin) Reductase in
Leuconostoc pseudomesenteroides
Authors: Rattray Fergal P. , Myling-Petersen Dorte , Larsen Dianna , Nilsson Dan
Published: January 2003

A plasmid-borne diacetyl (acetoin) reductase (
butA
) from
Leuconostoc pseudomesenteroides
CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the
butA
gene of
L. pseudomesenteroides
, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a
butA
mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in
L. pseudomesenteroides
. Plasmid-curing experiments demonstrated that the
butA
gene is carried on a 20-kb plasmid in
L. pseudomesenteroides
.
Effect of Raw-Milk Cheese Consumption on the Enterococcal Flora of Human Feces
Authors: Gelsomino Roberto , Vancanneyt Marc , Cogan Timothy M. , Swings Jean
Published: January 2003

Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. In the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of ∼125 g of a raw-milk Cheddar-type cheese containing 3.2 × 10
4
enterococci/g of cheese. Enterococcal counts ranged between 1.4 × 10
2
and 2.5 × 10
8
CFU/g of feces and differed from subject to subject and from week to week. The cheese contained mainly
Enterococcus casseliflavus
and a small population of
Enterococcus faecalis
. Clonal relationships were determined by pulsed-field gel electrophoresis. Before and after consumption of the cheese, samples from humans contained mainly
Enterococcus faecium
, with some of the clones being resident. During consumption of the cheese, one particular transient clone of
E. faecalis
, clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of
E. casseliflavus
from the cheese were also found in the feces of one of the subjects during cheese consumption. These results suggest that a clone need not be present in a food in high numbers to establish itself in the intestine.
Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and
mcrA
Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts
Authors: Lueders Tillmann , Friedrich Michael W.
Published: January 2003

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (
mcrA/mrtA
) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of
mcrA
-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.
Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons
Authors: Landeweert Renske , Leeflang Paula , Kuyper Thom W. , Hoffland Ellis , Rosling Anna , Wernars Karel , Smit Eric
Published: January 2003

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.
Bacillus anthracis
Diverges from Related Clades of the
Bacillus cereus
Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes
Authors: Cherif Ameur , Borin Sara , Rizzi Aurora , Ouzari Hadda , Boudabous Abdellatif , Daffonchio Daniele
Published: January 2003

Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the
Bacillus cereus
group showed that
B. anthracis
Davis TE702 and
B. mycoides
G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the
B. cereus
group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNA
Ile
and tRNA
Ala
) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNA
Ile
and tRNA
Ala
genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that
B. cereus
and
B. thuringiensis
represent a single clade. Three signature sequences discriminated
B. anthracis
from
B. cereus
and
B. thuringiensis
, indicating that the anthrax agent started evolving separately from the related clades of the
B. cereus
group.
B. mycoides
and
B. weienstephanensis
were very closely related, while
B. pseudomycoides
appeared the most distant species.
mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species
Authors: Brzostowicz Patricia C. , Walters Dana M. , Thomas Stuart M. , Nagarajan Vasantha , Rouvière Pierre E.
Published: January 2003

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In
Arthrobacter
sp. strain BP2 and
Rhodococcus
sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in
Rhodococcus
sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of
Arthrobacter
strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene
273:
305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in
Escherichia coli
.
Mitogen-Activated Protein Kinase in
Pfiesteria piscicida
and Its Growth Rate-Related Expression
Authors: Lin Senjie , Zhang Huan
Published: January 2003

A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate
Pfiesteria piscicida
. This cDNA (
Ppmapk1
) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40% amino acid similarity with MAPKs in other organisms. Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans. Using reverse transcription-PCR, expression of this gene was evaluated for
P. piscicida
cultures grown under different conditions. While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene,
Ppmapk1
expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism.
Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus
Authors: Nuanualsuwan Suphachai , Cliver Dean O.
Published: January 2003

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent.
Secretion of Active-Form
Streptoverticillium mobaraense
Transglutaminase by
Corynebacterium glutamicum
: Processing of the Pro-Transglutaminase by a Cosecreted Subtilisin-Like Protease from
Streptomyces albogriseolus
Authors: Kikuchi Yoshimi , Date Masayo , Yokoyama Kei-ichi , Umezawa Yukiko , Matsui Hiroshi
Published: January 2003

The transglutaminase secreted by
Streptoverticillium mobaraense
is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by
Corynebacterium glutamicum
when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from
C. glutamicum
. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by
C. glutamicum
but had no enzymatic activity. However, when the plasmid carrying the
S. mobaraense
transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from
Streptomyces albogriseolus
, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that
C. glutamicum
has potential as a host for industrial-scale protein production.
A Labeling Study To Elucidate the Biosynthesis of 4-(4-Hydroxyphenyl)-Butan-2-one (Raspberry Ketone) by
Nidula niveo-tomentosa
Authors: Zorn H. , Fischer-Zorn M. , Berger R. G.
Published: January 2003

Submerged cells of the basidiomycete
Nidula niveo-tomentosa
, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with
13
C-labeled
l
-phenylalanines and with [1-
13
C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-β-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.
Enhanced Heterologous Expression of Two
Streptomyces griseolus
Cytochrome P450s and
Streptomyces coelicolor
Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts
Authors: Hussain Haitham A. , Ward John M.
Published: January 2003

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of
Streptomyces griseolus
were expressed in
Escherichia coli
to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different
E. coli
expression hosts identified
E. coli
BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare
E. coli
tRNA species. The level of its expression was improved in the modified forms of
E. coli
BL21(DE3), which contain extra copies of rare codon
E. coli
tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from
Streptomyces coelicolor
downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in
Streptomyces lividans
1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160.
S. lividans
1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.
Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin fromLactobacillus plantarumNC8
Authors: Maldonado Antonio , Ruiz-Barba José Luis , Jiménez-Díaz Rufino
Published: January 2003

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated fromLactobacillus plantarumNC8 cultures which had been induced withLactococcus lactisMG1363 orPediococcus pentosaceusFBB63. This bacteriocin consists of two distinct peptides, named α and β, which were separated by C2-C18reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8α and PLNC8β, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (α) and 4,000 Da (β). The corresponding genes, designatedplNC8AandplNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature α and β peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream ofplNC8Band overlappingplNC8A. Upstream of the putative −35 region ofplNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.
Morphological and Phylogenetic Characterizations of FreshwaterThioplocaSpecies from Lake Biwa, Japan, and Lake Constance, Germany
Authors: Kojima Hisaya , Teske Andreas , Fukui Manabu
Published: January 2003

Filamentous, gliding, sulfide-oxidizing bacteria of the genusThioplocawere found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake BiwaThioplocaresembled morphologicallyThioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 μm; the diameters of completeThioplocafilaments ranged from 18 to 75 μm. The cell lengths ranged from 1.2 to 3.8 μm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of theThioplocafrom Lake Biwa was amplified by using newly designedThioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, theThioplocafrom Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake BiwaThioploca. Positive signals were obtained with the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake BiwaThioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, theThioplocafrom Lake Biwa and theThioplocafrom Lake Constance are closely related toT. ingricaand to each other.
Production of Optically Pure
d
-Lactic Acid in Mineral Salts Medium by Metabolically Engineered
Escherichia coli
W3110
Authors: Zhou Shengde , Causey T. B. , Hasona A. , Shanmugam K. T. , Ingram L. O.
Published: January 2003

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of
l
-lactic acid to
d
-lactic acid. Although the largest demand is for the
l
enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of
Escherichia coli
W3110 (prototrophic) as new biocatalysts for the production of
d
-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction.
d
-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this
d
-lactic acid was ∼98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (
frdABCD
), alcohol/aldehyde dehydrogenase (
adhE
), and pyruvate formate lyase (
pflB
). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (
ackA
). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of
d
-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.
Potential Rates of Fermentation in Digesta from the Gastrointestinal Tract of Pigs: Effect of Feeding Fermented Liquid Feed
Authors: Højberg Ole , Canibe Nuria , Knudsen Bettina , Jensen Bent Borg
Published: January 2003

Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system. The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota. In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum,
P
< 0.1; colon,
P
< 0.01; and rectum,
P
< 0.0001) of pigs fed FLF compared to pigs fed dry feed. No effect of diet was observed in the stomach (
P
= 0.71) or the distal part of the small intestine (
P
= 0.97). The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose. Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids. The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals. This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population. On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of
Escherichia coli
and
Salmonella
spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH. The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization. However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract. The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota.
Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci
Authors: Lunde Merete , Blatny Janet Martha , Lillehaug Dag , Aastveit Are Halvor , Nes Ingolf F.
Published: January 2003

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage φLC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six φLC3 lysogenic
Lactococcus lactis
subsp.
cremoris
host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the φLC3 prophage was found to vary with the growth phase of its host
L. lactis
IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the φLC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.
Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase
Authors: Kamerbeek Nanne M. , Olsthoorn Arjen J. J. , Fraaije Marco W. , Janssen Dick B.
Published: January 2003

The 4-hydroxyacetophenone monooxygenase (HAPMO) from
Pseudomonas fluorescens
ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant
Escherichia coli
, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1
R
,5
S
)-bicyclohept-2-en-6-one, with an enantiomeric ratio (
E
) of 20, thus enabling kinetic resolution to obtain the (1
S
,5
R
) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (
ee
) for the two lactones obtained [77%
ee
for (1
S
,5
R
)-2 and 34%
ee
for (1
R
,5
S
)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (
S
)-sulfoxides (
ee
> 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.
2-Hydroxypenta-2,4-dienoate Metabolic Pathway Genes in a Strong Polychlorinated Biphenyl Degrader,
Rhodococcus
sp. Strain RHA1
Authors: Sakai Masayuki , Miyauchi Keisuke , Kato Noboru , Masai Eiji , Fukuda Masao
Published: January 2003

A gram-positive polychlorinated biphenyl (PCB) degrader,
Rhodococcus
sp. strain RHA1, metabolizes biphenyl through the 2-hydroxypenta-2,4-dienoate (HPD) and benzoate metabolic pathways. The HPD metabolic pathway genes, the HPD hydratase (
bphE1
), 4-hydroxy-2-oxovalerate aldolase (
bphF1
), and acetaldehyde dehydrogenase (acylating) (
bphG
) genes, were cloned from RHA1. The deduced amino acid sequences of
bphGF1E1
have 30 to 58% identity with those of the HPD metabolic pathway genes of gram-negative bacteria. The order of these genes,
bphG
-
bphF1
-
bphE1
, differs from that of the HPD metabolic pathway genes,
bphE
-
bphG
-
bphF
, in gram-negative degraders of PCB, phenol, and toluene. Reverse transcription-PCR experiments indicated that the
bphGF1E1
genes are inducibly cotranscribed in cells grown on biphenyl and ethylbenzene. Primer extension analysis revealed that the transcriptional initiation site exists within the
bphR
gene located adjacent to and upstream of
bphG
, which is deduced to code a transcriptional regulator. The respective enzyme activities of
bphGF1E1
gene products were detected in
Rhodococcus erythropolis
IAM1399 carrying a
bphGF1E1
plasmid. The insertional inactivation of the
bphE1
,
bphF1
, and
bphG
genes resulted in the loss of the corresponding enzyme activities and diminished growth on both biphenyl and ethylbenzene. Severe growth interference was observed during growth on biphenyl. The growth defects were partially restored by the introduction of plasmids containing the respective intact genes. These results indicated that the cloned
bphGF1E1
genes are not only responsible for the primary metabolism of HPD during growth on both biphenyl and ethylbenzene but are also involved in preventing the accumulation of unexpected toxic metabolites, which interfere with the growth of RHA1.
Biodegradation of an Alicyclic Hydrocarbon by a Sulfate-Reducing Enrichment from a Gas Condensate-Contaminated Aquifer
Authors: Rios-Hernandez Luis A. , Gieg Lisa M. , Suflita Joseph M.
Published: January 2003

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera
Syntrophobacter
and
Desulfotomaculum
as well as a member of the
Cytophaga-Flexibacter-Bacteroides
group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.
Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis
Authors: Ennahar Saïd , Cai Yimin , Fujita Yasuhito
Published: January 2003

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera
Enterococcus
,
Lactobacillus
,
Lactococcus
,
Leuconostoc
,
Pediococcus
, and
Weissella
. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of
Lactobacillus plantarum
(24%),
Lactococcus lactis
(22%),
Leuconostoc pseudomesenteroides
(20%),
Pediococcus acidilactici
(11%),
Lactobacillus brevis
(11%),
Enterococcus faecalis
(7%),
Weissella kimchii
(3%), and
Pediococcus pentosaceus
(2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.
Compost-Induced Suppression of
Pythium
Damping-Off Is Mediated by Fatty-Acid-Metabolizing Seed-Colonizing Microbial Communities
Authors: McKellar Mary E. , Nelson Eric B.
Published: January 2003

Leaf composts were studied for their suppressive effects on
Pythium ultimum
sporangium germination, cottonseed colonization, and the severity of
Pythium
damping-off of cotton. A focus of the work was to assess the role of fatty-acid-metabolizing microbial communities in disease suppression. Suppressiveness was expressed within the first few hours of seed germination as revealed by reduced
P
.
ultimum
sporangium germination, reduced seed colonization, and reduced damping-off in transplant experiments. These reductions were not observed when cottonseeds were sown in a conducive leaf compost. Microbial consortia recovered from the surface of cottonseeds during the first few hours of germination in suppressive compost (suppressive consortia) induced significant levels of damping-off suppression, whereas no suppression was induced by microbial consortia recovered from cottonseeds germinated in conducive compost (conducive consortia). Suppressive consortia rapidly metabolized linoleic acid, whereas conducive consortia did not. Furthermore, populations of fatty-acid-metabolizing bacteria and actinobacteria were higher in suppressive consortia than in conducive consortia. Individual bacterial isolates varied in their ability to metabolize linoleic acid and protect seedlings from damping-off. Results indicate that communities of compost-inhabiting microorganisms colonizing cottonseeds within the first few hours after sowing in a
Pythium
-suppressive compost play a major role in the suppression of
P
.
ultimum
sporangium germination, seed colonization, and damping-off. Results further indicate that fatty acid metabolism by these seed-colonizing bacterial consortia can explain the
Pythium
suppression observed.
In Situ Exposure to Low Herbicide Concentrations Affects Microbial Population Composition and Catabolic Gene Frequency in an Aerobic Shallow Aquifer
Authors: de Lipthay Julia R. , Tuxen Nina , Johnsen Kaare , Hansen Lars H. , Albrechtsen Hans-Jørgen , Bjerg Poul L. , Aamand Jens
Published: January 2003

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 μg l
−1
) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations,
Pseudomonas
bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10
0
to 10
4
g
−1
sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes
tfdA
and
tfdB
(10
2
to 10
3
gene copies g
−1
sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of
tfd
genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of
Pseudomonas
bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4′,6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.
Online Monitoring of
Escherichia coli
Ghost Production
Authors: Haidinger W. , Szostak M. P. , Jechlinger W. , Lubitz W.
Published: January 2003

Controlled expression of cloned φX174 gene
E
in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.
Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Authors: Meroth Christiane B. , Walter Jens , Hertel Christian , Brandt Markus J. , Hammes Walter P.
Published: January 2003

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few
Lactobacillus
species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour),
Lactobacillus sanfranciscensis
and a new species,
L
.
mindensis
, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively
L
.
crispatus
and
L
.
pontis
became the predominant species in sourdough B and
L
.
crispatus
,
L
.
panis
, and
L
.
frumenti
became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran),
L
.
johnsonii
and
L
.
reuteri
were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which
L
.
fermentum
was also detected. Isolates of the species
L
.
sanfranciscensis
and
L
.
fermentum
were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.