bluuebunny/crossref_metadata_2025 huggingface dataset:
MARINATION OF SHEEP MUSCLES UNDER PRESSURE AND ITS EFFECT ON MEAT TEXTURE QUALITY
Authors: YASHODA K.P. , RAO R. JAGANNATHA , MAHENDRAKAR N.S. , RAO D. NARASIMHA
Published: July 2005

Marinated products are gaining importance in the market. Better penetration of salt(s) and/or acid(s) is desirable in marinated products. In order to improve ingredient penetration, experiments were carried out to marinate sheep thigh muscles under pressure. Semimembranosus and semitendinosus muscles from sheep, 4–5 years of age, were immersed (1 : 1) in a marinating solution consisting of 25 g citric acid and 200 g NaCl dissolved in 1 L of water (pH adjusted to 1.5). Right thigh muscles immersed in marinating solution were subjected to a pressure of 0.25, 0.50 or 0.75 kg/cm

2

(gauge) for 2 h at 27 ± 2C, while muscles from the left thigh were held at atmospheric pressure for 2 h (control). Marination under pressure reduced muscle pH 2–5%, increased titrable acidity 4–19%, increased salt content 15–45%, improved water‐holding capacity 10–21% and reduced shear values 24–48% depending on muscle and pressure applied. Thus, marination of muscles under pressure in the range of 0.25–0.75 kg/cm2enhances the penetration of salt and acid and improves texture quality of muscles.
Complexity in Strongly Correlated Electronic Systems
Authors: Dagotto Elbio
Published: July 2005

A wide variety of experimental results and theoretical investigations in recent years have convincingly demonstrated that several transition metal oxides and other materials have dominant states that are not spatially homogeneous. This occurs in cases in which several physical interactions—spin, charge, lattice, and/or orbital—are simultaneously active. This phenomenon causes interesting effects, such as colossal magnetoresistance, and it also appears crucial to understand the high-temperature superconductors. The spontaneous emergence of electronic nanometer-scale structures in transition metal oxides, and the existence of many competing states, are properties often associated with complex matter where nonlinearities dominate, such as soft materials and biological systems. This electronic complexity could have potential consequences for applications of correlated electronic materials, because not only charge (semiconducting electronic), or charge and spin (spintronics) are of relevance, but in addition the lattice and orbital degrees of freedom are active, leading to giant responses to small perturbations. Moreover, several metallic and insulating phases compete, increasing the potential for novel behavior.
A Self-Organized Vortex Array of Hydrodynamically Entrained Sperm Cells
Authors: Riedel Ingmar H. , Kruse Karsten , Howard Jonathon
Published: July 2005

Many patterns in biological systems depend on the exchange of chemical signals between cells. We report a spatiotemporal pattern mediated by hydrodynamic interactions. At planar surfaces, spermatozoa self-organized into dynamic vortices resembling quantized rotating waves. These vortices formed an array with local hexagonal order. Introducing an order parameter that quantifies cooperativity, we found that the array appeared only above a critical sperm density. Using a model, we estimated the hydrodynamic interaction force between spermatozoa to be ∼0.03 piconewtons. Thus, large-scale coordination of cells can be regulated hydrodynamically, and chemical signals are not required.
Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration
Authors: Dezawa Mari , Ishikawa Hiroto , Itokazu Yutaka , Yoshihara Tomoyuki , Hoshino Mikio , Takeda Shin-ichi , Ide Chizuka , Nabeshima Yo-ichi
Published: July 2005

Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.
Isolation of Two Seven-Membered Ring C
58
Fullerene Derivatives: C
58
F
17
CF
3
and C
58
F
18
Authors: Troshin Pavel A. , Avent Anthony G. , Darwish Adam D. , Martsinovich Natalia , Abdul-Sada Ala'a K. , Street Joan M. , Taylor Roger
Published: July 2005

Fluorination of C
60
at 550°C leads to milligram quantities of two stable fullerene derivatives with 58-carbon cage structures: C
58
F
18
and C
58
F
17
CF
3
. The compounds were characterized by mass spectrometry and fluorine nuclear magnetic resonance spectroscopy, and the data support a heptagonal ring in the framework. The resulting strain, which has hindered past attempts to prepare these smaller quasi-fullerenes, is mitigated here by hybridization change of some of the carbons in the pentagons from sp
2
to sp
3
because of fluorine addition. The loss of carbon from C
60
is believed to occur via sequential fluorine addition to a C–C single bond and an adjacent C[dbond]C bond, followed by loss of a :CF
2
carbene.
The Kinesin Klp2 Mediates Polarization of Interphase Microtubules in Fission Yeast
Authors: Carazo-Salas Rafael E. , Antony Claude , Nurse Paul
Published: July 2005

Fission yeast (
Schizosaccharomyces pombe
) cells grow longitudinally in a manner dependent on a polarized distribution of their interphase microtubules. We found that this distribution required sliding of microtubules toward the cell center along preexisting microtubules. This sliding was mediated by the minus end–directed kinesin motor Klp2, which helped microtubules to become properly organized with plus ends predominantly oriented toward the cell ends and minus ends toward the cell center. Thus, interphase microtubules in the fission yeast require motor activities for their proper organization.
Bovine Feces from Animals with Gastrointestinal Infections Are a Source of Serologically Diverse Atypical EnteropathogenicEscherichia coliand Shiga Toxin-ProducingE. coliStrains That Commonly Possess Intimin
Authors: Hornitzky Michael A. , Mercieca Kim , Bettelheim Karl A. , Djordjevic Steven P.
Published: July 2005

Shiga toxin-producingEscherichia coli(STEC) and enteropathogenicE. coli(EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR,E. colicells possessing combinations ofstx1,stx2,eae, andehxAwere detected by a combination of direct culture and enrichment inE. coli(EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin geneeaewas a feature of the STEC (35 [29.2%] of 120 isolates) and contrasted with the low prevalence (9 [0.5%] of 1,692 fecal samples possessed STEC witheae) of this gene among STEC recovered during extensive sampling of feces from healthy slaughter-age cattle in Australia (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Forty-seven STEC serotypes were identified, including O5:H−, O8:H19, O26:H−, O26:H11, O113:H21, O157:H7, O157:H− and Ont:H− which are known to cause severe disease in humans and 23 previously unreported STEC serotypes. Serotypes Ont:H− and O113:H21 represented the two most frequently isolated STEC isolates and were cultured from nine (4.7%) and seven (3.7%) animals, respectively. Fifteeneae-positiveE. coliserotypes, considered to represent atypical EPEC, were identified, with O111:H− representing the most prevalent. Using both techniques, STEC cells were cultured from 69 (36.1%) samples and EPEC cells were cultured from 30 (15.7%) samples, including 9 (4.7%) samples which yielded both STEC and EPEC. Culture on BVCC agar following enrichment in EC (modified) broth was the most successful method for the isolation of STEC (24.1% of samples), and direct culture on BVCC agar was the most successful method for the isolation of EPEC (14.1% samples). These studies show that diarrheagenic calves and cattle represent important reservoirs ofeae-positiveE. coli.
Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions
Authors: Wiles Siouxsie , Ferguson Kathryn , Stefanidou Martha , Young Douglas B. , Robertson Brian D.
Published: July 2005

The availability of cloned luciferase genes from fireflies (
luc
) and from bacteria (
luxAB
) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod
Gaussia princeps
(
Gluc
) has recently been cloned. We expressed the
Gluc
gene in
Mycobacterium smegmatis
using a shuttle vector and compared its performance with that of an existing
luxAB
reporter. In contrast to
luxAB
, the
Gluc
luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.
Quantitative Detection ofLegionella pneumophilain Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of thedotAGene
Authors: Yáñez M. A. , Carrasco-Serrano C. , Barberá V. M. , Catalán V.
Published: July 2005

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination ofLegionella pneumophilain water samples. Primers that amplify simultaneously an 80-bp fragment of thedotAgene fromL. pneumophilaand a recombinant fragment including a specific sequence of thegyrBgene fromAeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection ofL. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery ofL. pneumophilafrom different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification ofL. pneumophilain water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.
Polyketide Synthase Gene Responsible for Citrinin Biosynthesis in
Monascus purpureus
Authors: Shimizu Takeo , Kinoshita Hiroshi , Ishihara Shinji , Sakai Kanae , Nagai Shiro , Nihira Takuya
Published: July 2005

Citrinin produced by
Aspergillus
,
Penicillium
, and
Monascus
species is a polyketide compound that has nephrotoxic activity in mammals and is bactericidal toward gram-positive bacteria. To avoid the risk of citrinin contamination in other fermentation products produced by
Monascus purpureus
, knowledge of the citrinin biosynthetic genes is needed so that citrinin-nonproducing strains can be generated. We cloned a polyketide synthase (PKS) gene from
M. purpureus
with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene (
pksCT
) of 7,838 bp with a single 56-bp intron.
pksCT
encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase. There was no obvious thioesterase domain, which usually is downstream of the ACP domain in multi-aromatic-ring PKSs.
pksCT
transcription was correlated with citrinin production, suggesting that the
pksCT
gene product was involved in citrinin biosynthesis. Homologous recombination between the wild-type allele and a truncated disruption construct resulted in a
pksCT
-disrupted strain of
M. purpureus
. The disruptant did not produce citrinin, but a
pksCT
revertant generated by successive endogenous recombination events in the
pksCT
disruptant restored citrinin production, indicating that
pksCT
encoded the PKS responsible for citrinin biosynthesis in
M. purpureus
.
Anaerobic
n
-Alkane Metabolism by a Sulfate-Reducing Bacterium,
Desulfatibacillum aliphaticivorans
Strain CV2803
T
Authors: Cravo-Laureau Cristiana , Grossi Vincent , Raphel Danielle , Matheron Robert , Hirschler-Réa Agnès
Published: July 2005

The alkane-degrading, sulfate-reducing bacterium
Desulfatibacillum aliphaticivorans
strain CV2803
T
, recently isolated from marine sediments, was investigated for
n
-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of
D. aliphaticivorans
on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that
D. aliphaticivorans
strain CV2803
T
oxidizes
n
-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic
n
-alkane metabolism by
D. aliphaticivorans
is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.
Transcriptional Regulation and Characterization of a Novel β-Fructofuranosidase-Encoding Gene fromBifidobacterium breveUCC2003
Authors: Ryan Sinéad M. , Fitzgerald Gerald F. , van Sinderen Douwe
Published: July 2005

An operon involved in fructooligosaccharide breakdown was identified in the genome ofBifidobacterium breveUCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a β-fructofuranosidase. Active transcription of the operon was observed whenB. breveUCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The β-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37°C, respectively, and there was a dependence on bivalent cations, particularly manganese. TheKmandVmaxvalues for sucrose hydrolysis were determined to be 25 ± 2 mM and 24 ± 3 μmol min−1mg−1, respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the β(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the β(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria.
Description of Toluene Inhibition of Methyl Bromide Biodegradation in Seawater and Isolation of a Marine Toluene Oxidizer That Degrades Methyl Bromide
Authors: Goodwin Kelly D. , Tokarczyk Ryszard , Stephens F. Carol , Saltzman Eric S.
Published: July 2005

Methyl bromide (CH
3
Br) and methyl chloride (CH
3
Cl) are important precursors for destruction of stratospheric ozone, and oceanic uptake is an important component of the biogeochemical cycle of these methyl halides. In an effort to identify and characterize the organisms mediating halocarbon biodegradation, we surveyed the effect of potential cometabolic substrates on CH
3
Br biodegradation using a
13
CH
3
Br incubation technique. Toluene (160 to 200 nM) clearly inhibited CH
3
Br and CH
3
Cl degradation in seawater samples from the North Atlantic, North Pacific, and Southern Oceans. Furthermore, a marine bacterium able to co-oxidize CH
3
Br while growing on toluene was isolated from subtropical Western Atlantic seawater. The bacterium, Oxy6, was also able to oxidize
o
-xylene and the xylene monooxygenase (XMO) pathway intermediate 3-methylcatechol. Patterns of substrate oxidation, lack of acetylene inhibition, and the inability of the toluene 4-monooxygenase (T4MO)-containing bacterium
Pseudomonas mendocina
KR1 to degrade CH
3
Br ruled out participation of the T4MO pathway in Oxy6. Oxy6 also oxidized a variety of toluene (TOL) pathway intermediates such as benzyl alcohol, benzylaldehyde, benzoate, and catechol, but the inability of
Pseudomonas putida
mt-2 to degrade CH
3
Br suggested that the TOL pathway might not be responsible for CH
3
Br biodegradation. Molecular phylogenetic analysis identified Oxy6 to be a member of the family
Sphingomonadaceae
related to species within the
Porphyrobacter
genus. Although some
Sphingomonadaceae
can degrade a variety of xenobiotic compounds, this appears to be the first report of CH
3
Br degradation for this class of organism. The widespread inhibitory effect of toluene on natural seawater samples and the metabolic capabilities of Oxy6 indicate a possible link between aromatic hydrocarbon utilization and the biogeochemical cycle of methyl halides.
Degradation of Corn Fiber by
Clostridium cellulovorans
Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA
Authors: Koukiekolo Roger , Cho Hee-Yeon , Kosugi Akihiko , Inui Masayuki , Yukawa Hideaki , Doi Roy H.
Published: July 2005

Clostridium cellulovorans
, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of
C. cellulovorans
cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from
C. cellulovorans
may be more selective than random.
Previously Unknown Virus Infects Marine Diatom
Authors: Nagasaki Keizo , Tomaru Yuji , Takao Yoshitake , Nishida Kensho , Shirai Yoko , Suzuki Hidekazu , Nagumo Tamotsu
Published: July 2005

Diatoms are a major phytoplankton group that play important roles in maintaining oxygen levels in the atmosphere and sustaining the primary nutritional production of the aquatic environment. Among diatoms, the genus
Chaetoceros
is one of the most abundant and widespread. Temperature, climate, salinity, nutrients, and predators were regarded as important factors controlling the abundance and population dynamics of diatoms. Here we show that a viral infection can occur in the genus
Chaetoceros
and should therefore be considered as a potential mortality source.
Chaetoceros salsugineum
nuclear inclusion virus (CsNIV) is a 38-nm icosahedral virus that replicates within the nucleus of
C. salsugineum.
The latent period was estimated to be between 12 and 24 h, with a burst size of 325 infectious units per host cell. CsNIV has a genome structure unlike that of other viruses that have been described. It consists of a single molecule of covalently closed circular single-stranded DNA (ssDNA; 6,005 nucleotides), as well as a segment of linear ssDNA (997 nucleotides). The linear segment is complementary to a portion of the closed circle creating a partially double-stranded genome. Sequence analysis reveals a low but significant similarity to the replicase of circoviruses that have a covalently closed circular ssDNA genome. This new host-virus system will be useful for investigating the ecological relationships between bloom-forming diatoms and other viruses in the marine system. Our study supports the view that, given the diversity and abundance of plankton, the ocean is a treasury of undiscovered viruses.
Survey of Bovine Enterovirus in Biological and Environmental Samples by a Highly Sensitive Real-Time Reverse Transcription-PCR
Authors: Jiménez-Clavero Miguel Angel , Escribano-Romero Estela , Mansilla Carmen , Gómez Nuria , Córdoba Laura , Roblas Neftal , Ponz Fernando , Ley Victoria , Sáiz Juan-Carlos
Published: July 2005

Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination.
Amino Acid Catabolism by an
areA
-Regulated Gene Encoding an
l
-Amino Acid Oxidase with Broad Substrate Specificity in
Aspergillus nidulans
Authors: Davis Meryl A. , Askin Marion C. , Hynes Michael J.
Published: July 2005

The filamentous fungus
Aspergillus nidulans
can use a wide range of compounds as nitrogen sources. The synthesis of the various catabolic enzymes needed to breakdown these nitrogen sources is regulated by the
areA
gene, which encodes a GATA transcription factor required to activate gene expression under nitrogen-limiting conditions. The
areA102
mutation results in pleiotropic effects on nitrogen source utilization, including better growth on certain amino acids as nitrogen sources. Mutations in the
sarA
gene were previously isolated as suppressors of the strong growth of an
areA102
strain on
l
-histidine as a sole nitrogen source. We cloned the
sarA
gene by complementation of a
sarA
mutant and showed that it encodes an
l
-amino acid oxidase enzyme with broad substrate specificity. Elevated expression of this enzyme activity in an
areA102
background accounts for the strong growth of these strains on amino acids that are substrates for this enzyme. Loss of function
sarA
mutations, which abolish the
l
-amino acid oxidase activity, reverse the
areA102
phenotype. Growth tests with
areA102
and
sarA
mutants show that this enzyme is the primary route of catabolism for some amino acids, while other amino acids are metabolized through alternative pathways that yield either ammonium or glutamate for growth.
Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release
Authors: Kort Remco , O'Brien Andrea C. , van Stokkum Ivo H. M. , Oomes Suus J. C. M. , Crielaard Wim , Hellingwerf Klaas J. , Brul Stanley
Published: July 2005

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain
Bacillus subtilis
168 and from two food product isolates,
Bacillus subtilis
A163 and
Bacillus sporothermodurans
IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (
D
values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105°C, 120°C, and 131°C, respectively. The estimated
Z
values were 6.3°C, 6.1°C, and 9.7°C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (

T
c

), the temperature at which half the DPA content has been released within a fixed incubation time. We found

T
c

values for spores from
Bacillus
strains 168, A163, and IC4 of 108°C, 121°C, and 131°C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.
Relative Importance of Bacteriocin-Like Genes in Antagonism ofXanthomonas perforansTomato Race 3 toXanthomonas euvesicatoriaTomato Race 1 Strains
Authors: Hert A. P. , Roberts P. D. , Momol M. T. , Minsavage G. V. , Tudor-Nelson S. M. , Jones J. B.
Published: July 2005

In a previous study, tomato race 3 (T3) strains ofXanthomonas perforansbecame predominant in fields containing bothX. euvesicatoriaandX. perforansraces T1 and T3, respectively. This apparent ability to take over fields led to the discovery that there are three bacteriocin-like compounds associated with T3 strains. T3 strain 91-118 produces at least three different bacteriocin-like compounds (BCN-A, BCN-B, and BCN-C) antagonistic toward T1 strains. We determined the relative importance of the bacteriocin-like compounds by constructing the following mutant forms of a wild-type (WT) T3 strain to evaluate the antagonism to WT T1 strains: Mut-A (BCN-A−), Mut-B (BCN-B−), Mut-C (BCN-C−), Mut-AB, Mut-BC, and Mut-ABC. Although all mutant and WT T3 strains reduced the T1 populations in in planta growth room experiments, Mut-B and WT T3 were significantly more effective. Mutants expressing BCN-B and either BCN-A or BCN-C reduced T1 populations less than mutants expressing only BCN-A or BCN-C. The triple-knockout mutant Mut-ABC also had a significant competitive advantage over the T1 strain. In pairwise-inoculation field experiments where plants were coinoculated with an individual mutant or WT T3 strain and the T1 strain, the mutant strains and the WT T3 strain were reisolated from more than 70% of the lesions. WT T3 and Mut-B were the most frequently reisolated strains. In field experiments where plants were group inoculated with Mut-A, Mut-B, Mut-C, Mut-ABC, and WT T1 and T3 strains, Mut-B populations dominated all three seasons. In greenhouse and field experiments, the WT and mutant T3 strains had a selective advantage over T1 strains. Bacterial strains expressing both BCN-A and BCN-C appeared to have a competitive advantage over all other mutant and WT strains. Furthermore, BCN-B appeared to be a negative factor, with mutant T3 strains lacking BCN-B having a selective advantage in the field.
Diversity of Bacteria Growing in Natural Mineral Water after Bottling
Authors: Loy Alexander , Beisker Wolfgang , Meier Harald
Published: July 2005

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order
Burkholderiales
(6 OTUs) or the class
Alphaproteobacteria
(4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4′,6′-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that
Gammaproteobacteria
are the most important bacterial components of natural mineral water in bottles,
Betaproteobacteria
dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera
Hydrogenophaga
,
Aquabacterium
, and
Polaromonas
were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.
Degradation of Cyanophycin bySedimentibacter hongkongensisStrain KI andCitrobacter amalonaticusStrain G Isolated from an Anaerobic Bacterial Consortium
Authors: Obst Martin , Krug Andreas , Luftmann Heinrich , Steinbüchel Alexander
Published: July 2005

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacteriumSedimentibacter hongkongensisstrain KI as revealed by 16S rRNA analysis and the gram-negative enterobacteriumCitrobacter amalonaticusstrain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium.S. hongkongensisstrain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to β-Asp-Arg and β-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used byS. hongkongensisstrain KI, and significant growth on CGP did not occur. On the other hand,C. amalonaticusstrain G did not degrade CGP but grew on the β-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity ofS. hongkongensisstrain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.
Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy
Streptococcus thermophilus
Strains Reveals Four Different Fermentation Profiles
Authors: de Vin Filip , Rådström Peter , Herman Lieve , De Vuyst Luc
Published: July 2005

Lactose-limited fermentations of 49 dairy
Streptococcus thermophilus
strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the
gal
promoter seems to play an important role in the galactose phenotype, the
galR-galK
intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The
gal
promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six
S. thermophilus
strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their
gal
genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different
S. thermophilus
strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their
galR-galK
intergenic region were closely related.
pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon
Authors: Walker Alan W. , Duncan Sylvia H. , McWilliam Leitch E. Carol , Child Matthew W. , Flint Harry J.
Published: July 2005

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes.Bacteroideslevels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producingRoseburiagroup were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, threeBacteroidesspecies grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. TwoRoseburiaisolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth ofBacteroidesspp.
Identification of a Positive Transcription Regulatory Element within the Coding Region of the
nifLA
Operon in
Azotobacter vinelandii
Authors: Mitra Ranjana , Das Hirendra K. , Dixit Aparna
Published: July 2005

Nitrogen fixation in
Azotobacter vinelandii
is regulated by the
nifLA
operon. NifA activates the transcription of
nif
genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the
nifLA
operon of
A. vinelandii
itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the
nifLA
operon, showing it to be similar to the σ
54
-dependent promoters. We also identified a positive
cis-
acting regulatory element (+134 to +790) of the
nifLA
operon within the coding region of the
nifL
gene of
A. vinelandii
. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the
nifLA
promoter activity. The absence of NtrC-like binding sites in the upstream region of the
nifLA
operon in
A. vinelandii
makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in
nifLA
regulation in
A. vinelandii
.
Dehalogenation of the Herbicides Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by
Desulfitobacterium chlororespirans
Authors: Cupples Alison M. , Sanford Robert A. , Sims Gerald K.
Published: July 2005

Desulfitobacterium chlororespirans
has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of
ortho
chlorines on polysubstituted phenols. Here, we examine the ability of
D. chlororespirans
to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 μM) and DBHB (250 to 260 μM) were used as electron acceptors for growth. Doubling times for growth (means ± standard deviations for triplicate cultures) on bromoxynil (18.4 ± 5.2 h) and DBHB (11.9 ± 1.4 h), determined by rate of [
14
C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the
Desulfitobacterium
genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of
D. chlororespirans
.
Hyaluronic Acid Production in
Bacillus subtilis
Authors: Widner Bill , Behr Régine , Von Dollen Steve , Tang Maria , Heu Tia , Sloma Alan , Sternberg Dave , DeAngelis Paul L. , Weigel Paul H. , Brown Steve
Published: July 2005

The
hasA
gene from
Streptococcus equisimilis
, which encodes the enzyme hyaluronan synthase, has been expressed in
Bacillus subtilis
, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the
hasA
gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in
B. subtilis
and that overexpressing the
hasA
gene along with the endogenous
tuaD
gene is sufficient for high-level production of HA. In addition, the
B. subtilis
-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.
Empirical Establishment of Oligonucleotide Probe Design Criteria
Authors: He Zhili , Wu Liyou , Li Xingyuan , Fields Matthew W. , Zhou Jizhong
Published: July 2005

Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of ≤85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (≤15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than −30 kcal/mol for 50-mer probes or −40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.
Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by
Archaeoglobus fulgidus
Strain Z
Authors: Habicht Kirsten S. , Salling Lilian , Thamdrup Bo , Canfield Donald E.
Published: July 2005

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for
Archaeoglobus fulgidus
strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO
2
. This is the first time that the production of formate has been reported for
A. fulgidus
. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO
2
produced at high concentrations. We suggest different pathways for lactate oxidation by
A. fulgidus
at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.
Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study
Authors: Hendrickx Barbara , Dejonghe Winnie , Boënne Wesley , Brennerova Maria , Cernik Miroslav , Lederer Tomas , Bucheli-Witschel Margarete , Bastiaens Leen , Verstraete Willy , Top Eva M. , Diels Ludo , Springael Dirk
Published: July 2005

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with
tmoA
- and
xylM/xylE1
-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only
tmoA
-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of
Proteobacteria
. At the uncontaminated location,
Actinobacteria
and
Proteobacteria
were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.