bluuebunny/crossref_metadata_2025 huggingface dataset:
Separate Sensory Pathways for Photomorphogenesis in Phycomyces
Authors: Flores Rafael , Cerdá‐Olmedo Enrique , Corrochano Luis M.
Published: January 2008

The zygomycete Phycomyces blakesleeanus develops two types of sporangiophores of very different size: macrophores and microphores. Blue light inhibits the development of microphores and stimulates the development of macrophores. These responses are called photomicrophorogenesis and photomacrophorogenesis, respectively; phototropism designates the growth of the macrophores toward blue light and photocarotenogenesis, the increased accumulation of β‐carotene under blue light. We have isolated three pim mutants that develop microphores under continuous illumination at fluence rates that inhibit them in the wild type. The thresholds for photomicrophorogenesis in the mutants were higher than in the wild type, but the thresholds for photomacrophorogenesis and phototropism did not change. At least one of the pim mutants had a much higher threshold for photocarotenogenesis than the wild type. A madJ mutant strain, already known for its very defective phototropism, was defective for photomicrophorogenesis, but normal for photomacrophorogenesis and photocarotenogenesis. The requirement for the pim and madJ gene functions separates the two photomorphogenetic responses. The results indicate that the four responses to blue light are based on a combinatorial use of sensory transducers.
Origin and propagation of individual slow waves along the intact feline small intestine
Authors: Lammers Wim J. E. P. , Stephen Betty
Published: December 2007

The pattern of propagation of slow waves in the small intestine is not clear. Specifically, it is not known whether propagation is determined by a single dominant ICC‐MP (Interstitial cells of Cajal located in the Myenteric Plexus) pacemaker unit or whether there are multiple active pacemakers. To determine this pattern of propagation, waveforms were recorded simultaneously from 240 electrodes distributed along the whole length of the intact isolated feline small intestine. After the experiments, the propagation patterns of successive individual slow waves were analysed. In the intact small intestine, there was only a single slow wave pacemaker unit active, and this was located at or 6–10 cm from the pyloric junction. From this site, slow waves propagated in the aboral direction at gradually decreasing velocities. The majority of slow waves (73%) reached the ileocaecal junction while the remaining waves were blocked. Ligation of the intestine at one to four locations led to: (a) decrease in the distal frequencies; (b) disappearance of distal propagation blocks; (c) increase in velocities; (d) emergence of multiple and unstable pacemaker sites; and (e) propagation from these sites in the aboral and oral directions. In conclusion, in the quiescent feline small intestine a single pacemaker unit dominates the organ, with occasional propagation blocks of the slow waves, thereby producing the well‐known frequency gradient.
Long‐term effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki rat
Authors: Howarth Frank C. , Jacobson Michael , Shafiullah Mohamed , Adeghate Ernest
Published: December 2007

In vivo biotelemetry studies have demonstrated a variety of heart rhythm disturbances in type 1 diabetes mellitus. In the streptozotocin (STZ)‐induced diabetic rat, these disturbances have included reductions in heart rate (HR) and heart rate variability (HRV) and an electrocardiogram that displays prolonged QRS duration and Q–T interval. The aim of this study was to investigate the chronic effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki (GK) rat. Transmitter devices were surgically implanted in the peritoneal cavity of young male GK and age‐matched Wistar control rats. Electrodes from the transmitter were arranged in Einthoven bipolar lead II configuration. Electrocardiogram, physical activity and body temperature data were recorded in rats from age 2 to 15 months. Data were acquired for 2 weeks each month. Non‐fasting blood glucose, glucose tolerance and body weight were measured periodically. In GK rats, growth rate and maximal attained body weight were significantly reduced and non‐fasting blood glucose was progressively increased compared with age‐matched Wistar control animals. Heart rate was significantly lower in GK compared with control rats at 2, 7 and 15 months of age. At 2 months of age, HR was 316 ± 6 beats min−1 in GK rats compared with 370 ± 7 beats min−1 in Wistar control animals. There was a progressive age‐dependent decline in HRV in Wistar control rats; however, HRV in GK rats did not alter significantly with age. Heart rate variability was significantly reduced in GK compared with Wistar control rats at 2 and 7 months. At 2 months of age, HRV was 28 ± 2 beats min−1 in GK rats compared with 38 ± 3 beats min−1 in Wistar control rats. Reduced HR in GK rats may be an inherited characteristic. The absence of age‐dependent reductions in HRV in GK rats may be a consequence of an underlying impairment of autonomic control which manifests at early age.
Genetically altered animal models for Mas and angiotensin‐(1–7)
Authors: Alenina Natalia , Xu Ping , Rentzsch Brit , Patkin Eugene L. , Bader Michael
Published: December 2007

Mas is the receptor for angiotensin‐(1–7) and is involved in cardiovascular and neuronal regulation, in which the heptapeptide also plays a major role. Mas‐deficient mice have been generated by us, and their characterization has shown that Mas has important functions in behaviour and cardiovascular regulation. These mice exhibit increased anxiety but, despite an enhanced long‐term potentiation in the hippocampus, do not perform better in learning experiments. When Mas‐deficient mice are backcrossed to the FVB/N genetic background, a cardiovascular phenotype is uncovered, in that the backcrossed animals become hypertensive. Concordant with our detection by fluorescent in situ hybridization of Mas mRNA in mouse endothelium, this phenotype is caused by endothelial dysfunction based on a dysbalance between nitric oxide and reactive oxygen species in the vessel wall. In agreement with these data, transgenic spontaneously hypertensive stroke‐prone rats overexpressing ACE2 in the vessel wall exhibit reduced blood pressure as a result of improved endothelial function. Moreover, angiotensin‐(1–7) overexpression in transgenic rats has cardioprotective and haemodynamic effects. In conclusion, the angiotensin‐(1–7)–Mas axis has important functional implications for vascular regulation and blood pressure control, particularly in pathophysiological situations.
Alterations in in vitro function and protein oxidation of rat sarcoplasmic reticulum Ca2+‐ATPase during recovery from high‐intensity exercise
Authors: Matsunaga Satoshi , Mishima Takaaki , Yamada Takashi , Inashima Shuichiro , Wada Masanobu
Published: December 2007

The hypothesis tested in this study was that the extent to which sarcoplasmic reticulum (SR) Ca2+‐ATPase is oxidized would correlate with a decline in its activity. For this purpose, changes in the SR Ca2+‐sequestering ability and the contents of carbonyl and sulfhydryl groups during recovery after exercise were examined in the superficial portions of vastus lateralis muscles from rats subjected to 5 min running at an intensity corresponding to maximal oxygen uptake (50 m min−1, 10% gradient). A single bout of exercise elicited a 22.4% reduction (P < 0.05) in SR Ca2+‐ATPase activity. The decreased activity progressively reverted to normal levels during recovery after exercise, reaching normal levels after 60 min of recovery. This change was paralleled by a depressed SR Ca2+‐uptake rate, and the proportional alteration in these two variables resulted in no change in the ratio of Ca2+‐uptake rate to Ca2+‐ATPase activity. The contents of SR Ca2+‐ATPase protein and sulfhydryl groups in microsomes were unchanged after exercise and during recovery periods. In contrast, the content of carbonyl groups in SR Ca2+‐ATPase behaved in an opposite manner to that of SR Ca2+‐ATPase activity. An approximately 80% augmentation (P < 0.05) in the carbonyl group content occurred immediately after exercise. The elevated carbonyl content decreased towards normal levels during 60 min of recovery. These results are strongly suggestive that oxidation of SR Ca2+‐ATPase is responsible, at least in part, for a decay in the SR Ca2+‐pumping function produced by high‐intensity exercise and imply that oxidized proteins may be repaired during recovery from exercise.
Transformation of adult rat cardiac myocytes in primary culture
Authors: Banyasz Tamas , Lozinskiy Ilya , Payne Charles E. , Edelmann Stephanie , Norton Byron , Chen Biyi , Chen‐Izu Ye , Izu Leighton T. , Balke C. William
Published: December 2007

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long‐term cell culture. Morphometric parameters, sarcomere length, T‐tubule density, cell capacitance, L‐type calcium current (ICa,L), inward rectifier potassium current (IK1), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 ± 70 μm2 on day 0 to 1432 ± 50 μm2 on day 5), T‐tubule density, systolic shortening (from 0.11 ± 0.02 to 0.05 ± 0.01 μm) and amplitude of calcium transients (from 1.54 ± 0.19 to 0.67 ± 0.19) over 5 days of culture. The negative force–frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 ± 8 to 105 ± 11 pF) and membrane currents were also reduced (ICa,L, from 3.98 ± 0.39 to 2.12 ± 0.37 pA pF; and IK1, from 34.34p ± 2.31 to 18.00 ± 5.97 pA pF−1). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 ± 2.5 to 34.2 ± 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.
Time‐dependent changes in caspase‐3 activity and heat shock protein 25 after spinal cord transection in adult rats
Authors: Huey Kimberly A. , Roy Roland R. , Zhong Hui , Lullo Carolyn
Published: December 2007

Chronic reductions in muscle activation and loading are associated with decreased heat shock protein 25 (Hsp25) expression and phosphorylation (pHsp25) which, in turn, may contribute to elevated caspase‐3‐mediated muscle protein breakdown. Thus, the purpose of the present study was to determine whether there are any changes in Hsp25, pHsp25 and caspase‐3 activity among rat muscles having different fibre type compositions and functions [soleus, adductor longus (AL), plantaris and tibialis anterior (TA)] at 0 (control), 1, 8 or 28 days after a complete spinal cord transection (ST). The Hsp25 levels were unaffected on days 1 and 8 in all muscles, except for a significant reduction on day 8 in plantaris. The Hsp25 levels were lower than control values in all muscles except TA on day 28. The pHsp25 levels were lower than control values after 8 and 28 days in plantaris and AL and after 28 days in soleus, but higher than control in TA after 8 and 28 days. Caspase‐3 activity was higher in ST than control rats on day 8 in all muscles except TA. Caspase‐3 activity was negatively correlated with muscle mass for all muscles. In plantaris, Hsp25 and pHsp25 were negatively correlated with caspase‐3 activity and Hsp25 was correlated with muscle mass. These relationships were not observed in other muscles. Thus, the effects of ST on Hsp25 and caspase‐3 are muscle specific and time dependent, factors that should be considered in developing any intervention to maintain muscle mass after a spinal cord injury.
Risk of Gastrointestinal Disease Associated with Exposure to Pathogens in the Water of the Lower Passaic River
Authors: Donovan Ellen , Unice Ken , Roberts Jennifer D. , Harris Mark , Finley Brent
Published: December 2007

During precipitation events, untreated human sewage is often intentionally discharged to surface water bodies via combined sewer overflow (CSO) systems in order to avoid overloading wastewater treatment plants. The purpose of this analysis was to evaluate the risk of pathogen-related disease associated with CSO discharges into the Lower Passaic River. Concentrations of fecal coliform, total coliform, fecal
Streptococcus
, and fecal
Enterococcus
bacteria were measured at six river locations on six different days in 2003 (
n
= 36). In addition, water samples (
n
= 2) were collected directly from and in the immediate vicinity of a discharging CSO in Newark, NJ. These samples were analyzed for fecal coliforms, total coliforms, fecal
Streptococcus
, fecal
Enterococcus
,
Giardia lamblia
,
Cryptosporidium parvum
, and several viruses. Risk estimates for gastrointestinal illness and
Giardia
infection resulting from indirect and direct ingestion of contaminated water were calculated for three potential exposure scenarios: visitor, recreator, and homeless person. Single-event risk was first evaluated for the three individual exposure scenarios; overall risk was then determined over a 1-year period. Monte Carlo techniques were used to characterize uncertainty. Nearly all of the pathogen concentrations measured in the Passaic River exceeded health-based water quality criteria and in some cases were similar to levels reported for raw sewage. The probability of contracting gastrointestinal illness due to fecal
Streptococcus
and
Enterococcus
from incidental ingestion of water over the course of a year ranged from 0.14 to nearly 0.70 for the visitor and recreator scenarios, respectively. For the homeless person exposure scenario, the risk for gastrointestinal illness reached 0.88 for fecal
Streptococcus
and
Enterococcus
, while the probability of
Giardia
infection was 1.0. This risk analysis suggests that, due to the levels of pathogens present in the Lower Passaic River, contact with the water poses, and will continue to pose, significant human health risks until CSO discharges are adequately controlled or abated.
Blackbirds and Song Thrushes Constitute a Key Reservoir of
Borrelia garinii
, the Causative Agent of Borreliosis in Central Europe
Authors: Taragel'ová Veronika , Koči Juraj , Hanincová Klára , Kurtenbach Klaus , Derdáková Markéta , Ogden Nick H. , Literák Ivan , Kocianová Elena , Labuda Milan
Published: December 2007

Blackbirds (
Turdus merula
) and song thrushes (
Turdus philomelos
) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as
Borrelia garinii
and
Borrelia valaisiana
. We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.
Contribution of Citrate Metabolism to the Growth of
Lactococcus lactis
CRL264 at Low pH
Authors: Sánchez Claudia , Neves Ana Rute , Cavalheiro João , dos Santos Margarida Moreira , García-Quintáns Nieves , López Paloma , Santos Helena
Published: December 2007

Lactococcus lactis
subsp.
lactis
biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo
13
C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions.
Risk of Gastrointestinal Disease Associated with Exposure to Pathogens in the Sediments of the Lower Passaic River
Authors: Donovan E. P. , Staskal D. F. , Unice K. M. , Roberts J. D. , Haws L. C. , Finley B. L. , Harris M. A.
Published: December 2007

High levels of pathogenic microorganisms have been documented previously in waters of the Lower Passaic River in northern New Jersey. The purpose of this study was to characterize the microbial contamination of river sediments near combined sewer overflows (CSOs), a known source of pathogens. Concentrations of fecal coliform, total coliform, fecal
Streptococcus
, fecal
Enterococcus
,
Pseudomonas aeruginosa
,
Staphylococcus aureus
,
Giardia lamblia
, and
Cryptosporidium parvum
organisms were measured in 16 samples from three mudflat locations along the Lower Passaic River, as well as from an upstream location. Selected samples were also analyzed for antibiotic resistance. All of the samples contained high concentrations of total coliform, fecal coliform, fecal
Streptococcus
, and fecal
Enterococcus
organisms. Analysis of isolates of
Staphylococcus aureus
,
Pseudomonas aeruginosa
, and
Escherichia coli
from several samples indicated that each strain was resistant to at least one antibiotic typically used in clinical settings. Eight of 16 samples contained
Giardia
, and one sample contained
Cryptosporidium
. With these sampling data, a quantitative microbial risk assessment was conducted to evaluate the probability of infection or illness resulting from incidental ingestion of contaminated sediments over a 1-year period. Three potential exposure scenarios were considered: visitor, recreator, and homeless person. Single-event risk was first evaluated for the three individual exposure scenarios; overall risk was then determined over a 1-year period using Monte Carlo techniques to characterize uncertainty. For fecal
Streptococcus
and
Enterococcus
, annualized risk estimates for gastrointestinal illness ranged from approximately 0.42 to 0.53 for recreators, 0.07 to 0.10 for visitors, and 0.62 to 0.72 for homeless individuals across the three sampling locations. Annualized risk of
Giardia
infection ranged from 0.14 to 0.64 for recreators, 0.01 to 0.1 for visitors, and 0.30 to 0.87 for homeless individuals, across all locations where detected.
Cryptosporidium
was detected at one location, and the corresponding annualized risk of infection was 0.32, 0.05, and 0.51 for recreators, visitors, and homeless individuals, respectively. This risk assessment suggests that pathogen-contaminated sediments near areas of CSO discharge in the Lower Passaic River could pose a health risk to individuals coming into contact with sediments in the mudflat areas.
Enrichment, Isolation, and Phylogenetic Identification of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria from Elizabeth River Sediments
Authors: Hilyard Edward J. , Jones-Meehan Joanne M. , Spargo Barry J. , Hill Russell T.
Published: December 2007

The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of
14
C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.
Extracellular Expression of a Functional Recombinant
Ganoderma lucidium
Immunomodulatory Protein by
Bacillus subtilis
and
Lactococcus lactis
Authors: Yeh Chuan M. , Yeh Chun K. , Hsu Xun Y. , Luo Qiu M. , Lin Ming Y.
Published: December 2007

Bacillus subtilis
and
Lactococcus lactis
are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel
Ganoderma lucidium
immunomodulatory protein LZ-8, recombinant
LZ-8
, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SP
YaB
), recombinant LZ-8 was expressed extracellularly in
Bacillus subtilis
and
Lactococcus lactis
. In the absence of SP
YaB
, recombinant LZ-8 was expressed extracellularly in
B. subtilis
, but not in
L. lactis
. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.
Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading
Sphingomonas
sp. Strain LB126
Authors: Schuler Luc , Ní Chadhain Sinéad M. , Jouanneau Yves , Meyer Christine , Zylstra Gerben J. , Hols Pascal , Agathos Spiros N.
Published: December 2007

In this study, the genes involved in the initial attack on fluorene by
Sphingomonas
sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader
Terrabacter
sp. strain DBF63, were identified. When overexpressed in
Escherichia coli
, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-
p
-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from
Terrabacter
sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.
Intergeneric Coaggregation among Drinking Water Bacteria: Evidence of a Role for
Acinetobacter calcoaceticus
as a Bridging Bacterium
Authors: Simões Lúcia Chaves , Simões Manuel , Vieira Maria João
Published: December 2007

Intergeneric coaggregation of drinking water bacteria was tested.
Acinetobacter calcoaceticus
was found not only to autoaggregate but also to coaggregate with four of the five other isolates (
Burkholderia cepacia
,
Methylobacterium
sp.,
Mycobacterium mucogenicum
,
Sphingomonas capsulata
, and
Staphylococcus
sp.). In its absence, no coaggregation was found. Interactions were lectin-saccharide mediated. The putative bridging function of
A. calcoaceticus
was evidenced by multispecies biofilm studies, through a strain exclusion process.
Predictive Model for Inactivation of Feline Calicivirus, a Norovirus Surrogate, by Heat and High Hydrostatic Pressure
Authors: Buckow Roman , Isbarn Sonja , Knorr Dietrich , Heinz Volker , Lehmacher Anselm
Published: December 2007

Noroviruses, which are members of the
Caliciviridae
family, represent the leading cause of nonbacterial gastroenteritis in developed countries; such norovirus infections result in high economic costs for health protection. Person-to-person contact, contaminated water, and foods, especially raw shellfish, vegetables, and fruits, can transmit noroviruses. We inactivated feline calicivirus, a surrogate for the nonculturable norovirus, in cell culture medium and mineral water by heat and high hydrostatic pressure. Incubation at ambient pressure and 75°C for 2 min as well as treatment at 450 MPa and 15°C for 1 min inactivated more than 7 log
10
PFU of calicivirus per ml in cell culture medium or mineral water. The heat and pressure time-inactivation curves obtained with the calicivirus showed tailing in the logarithmic scale. Modeling by
n
th-order kinetics of the virus inactivation was successful in predicting the inactivation of the infective virus particles. The developed model enables the prediction of the calicivirus reduction in response to pressures up to 500 MPa, temperatures ranging from 5 to 75°C, and various treatment times. We suggest high pressure for processing of foods to reduce the health threat posed by noroviruses.
Functional-Genomics-Based Identification and Characterization of Open Reading Frames Encoding α-Glucoside-Processing Enzymes in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Comfort Donald A. , Chou Chung-Jung , Conners Shannon B. , VanFossen Amy L. , Kelly Robert M.
Published: December 2007

Bioinformatics analysis and transcriptional response information for
Pyrococcus furiosus
grown on α-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 β-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative α-glucan-processing enzymes.
Contribution of Copper Ion Resistance to Survival of
Escherichia coli
on Metallic Copper Surfaces
Authors: Santo Christophe Espírito , Taudte Nadine , Nies Dietrich H. , Grass Gregor
Published: December 2007

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of
Escherichia coli
on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.
Genetic Diversity of Hydrogen-Producing Bacteria in an Acidophilic Ethanol-H2-Coproducing System, Analyzed Using the [Fe]-Hydrogenase Gene
Authors: Xing Defeng , Ren Nanqi , Rittmann Bruce E.
Published: December 2007

Hydrogen gas (H2) produced by bacterial fermentation of biomass can be a sustainable energy source. The ability to produce H2gas during anaerobic fermentation was previously thought to be restricted to a few species within the generaClostridiumandEnterobacter. This work reports genomic evidence for the presence of novel H2-producing bacteria (HPB) in acidophilic ethanol-H2-coproducing communities that were enriched using molasses wastewater. The majority of the enriched dominant populations in the acidophilic ethanol-H2-coproducing system were affiliated with low-G+C-content gram-positive bacteria,Bacteroidetes, andActinobacteria, based on the 16S rRNA gene. However, PCR primers designed to specifically target bacterialhydAyielded 17 uniquehydAsequences whose amino acid sequences differed from those of known HPB. The putative ethanol-H2-coproducing bacteria comprised 11 novel phylotypes closely related toEthanoligenens harbinense,Clostridium thermocellum, andClostridium saccharoperbutylacetonicum. Furthermore, analysis of the alcohol dehydrogenase isoenzyme also pointed to anE. harbinense-like organism, which is known to have a high conversion rate of carbohydrate to H2and ethanol. We also found six novel HPB that were associated with lactate-, propionate-, and butyrate-oxidizing bacteria in the acidophilic H2-producing sludge. Thus, the microbial ecology of mesophilic and acidophilic H2fermentation involves many other bacteria in addition toClostridiumandEnterobacter.
Adherence of
Helicobacter pylori
to Abiotic Surfaces Is Influenced by Serum
Authors: Williams John C. , McInnis Karla A. , Testerman Traci L.
Published: December 2007

Helicobacter pylori
bacteria cultured in a chemically defined medium without serum readily adhere to a variety of abiotic surfaces. Growth produces microcolonies that spread to cover the entire surface, along with a planktonic subpopulation. Serum inhibits adherence. Initial attachment is protein mediated, but other molecules are responsible for more permanent attachment.
System Using Tandem Repeats of the cA Peptidoglycan-Binding Domain from
Lactococcus lactis
for Display of both N- and C-Terminal Fusions on Cell Surfaces of Lactic Acid Bacteria
Authors: Okano Kenji , Zhang Qiao , Kimura Sakurako , Narita Junya , Tanaka Tsutomu , Fukuda Hideki , Kondo Akihiko
Published: December 2007

Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from
Lactococcus lactis
). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and α-amylase from
Streptococcus bovis
148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of
L. lactis
cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of α-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of
Lactobacillus plantarum
and
Lactobacillus casei
.
Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by
Shewanella
spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction
Authors: Pinchuk Grigoriy E. , Ammons Christine , Culley David E. , Li Shu-Mei W. , McLean Jeff S. , Romine Margaret F. , Nealson Kenneth H. , Fredrickson Jim K. , Beliaev Alexander S.
Published: December 2007

The solubility of orthophosphate (PO
4
3−
) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus
Shewanella. Shewanella oneidensis
MR-1,
Shewanella putrefaciens
CN32, and
Shewanella
sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca
2+
-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca
2+
for growth with DNA. For
S. oneidensis
MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca
2+
-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.
The Inhibitory Spectrum of Thermophilin 9 from
Streptococcus thermophilus
LMD-9 Depends on the Production of Multiple Peptides and the Activity of BlpG
St
, a Thiol-Disulfide Oxidase
Authors: Fontaine Laetitia , Hols Pascal
Published: December 2007

The
blp
St
cluster of
Streptococcus thermophilus
LMD-9 was recently shown to contain all the genetic information required for the production of bacteriocins active against other
S. thermophilus
strains. In this study, we further investigated the antimicrobial activity of
S. thermophilus
LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as
Listeria monocytogenes
. Using deletion mutants, we investigated the contribution of the three putative bacteriocin-encoding operons
blpD
St
-
orf2
,
blpU
St
-
orf3
, and
blpE
St
-
blpF
St
(
bac
St
operons) and of the
blpG
St
gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the
blp
St
locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the
bac
St
operons, BlpD
St
alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The
blpD
St
gene forms an operon with its associated immunity gene(s), and this functional bacteriocin/immunity module could easily be transferred to
Lactococcus lactis
. The remaining three Bac
St
peptides, BlpU
St
, BlpE
St
, and BlpF
St
, confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The
blpG
St
gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most
Listeria
species. By complementation of the motility deficiency of
Escherichia coli dsbA
, we showed that
blpG
St
encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.
Changes in Bacterial Communities of the Marine Sponge
Mycale laxissima
on Transfer into Aquaculture
Authors: Mohamed Naglaa M. , Enticknap Julie J. , Lohr Jayme E. , McIntosh Scott M. , Hill Russell T.
Published: December 2007

The changes in bacterial communities associated with the marine sponge
Mycale laxissima
on transfer to aquaculture were studied using culture-based and molecular techniques.
M. laxissima
was maintained alive in flowthrough and closed recirculating aquaculture systems for 2 years and 1 year, respectively. The bacterial communities associated with wild and aquacultured sponges, as well as the surrounding water, were assessed using 16S rRNA gene clone library analysis and denaturing gradient gel electrophoresis (DGGE). Bacterial richness and diversity were measured using DOTUR computer software, and clone libraries were compared using S-LIBSHUFF. DGGE analysis revealed that the diversity of the bacterial community of
M. laxissima
increased when sponges were maintained in aquaculture and that bacterial communities associated with wild and aquacultured
M. laxissima
were markedly different than those of the corresponding surrounding water. Clone libraries of bacterial 16S rRNA from sponges confirmed that the bacterial communities changed during aquaculture. These communities were significantly different than those of seawater and aquarium water. The diversity of bacterial communities associated with
M. laxissima
increased significantly in aquaculture. Our work shows that it is important to monitor changes in bacterial communities when examining the feasibility of growing sponges in aquaculture systems because these communities may change. This could have implications for the health of sponges or for the production of bioactive compounds by sponges in cases where these compounds are produced by symbiotic bacteria rather than by the sponges themselves.
Fermentative Utilization of Glycerol by
Escherichia coli
and Its Implications for the Production of Fuels and Chemicals
Authors: Murarka Abhishek , Dharmadi Yandi , Yazdani Syed Shams , Gonzalez Ramon
Published: December 2007

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in
Escherichia coli
had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h
−1
. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-
13
C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-
13
C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (∼95%) and redox (∼96%) balances. On the other hand, cultivation conditions that prevent H
2
accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H
2
is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.
Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment
Authors: Xiao Zhizhuang , Bergeron Hélène , Grosse Stephan , Beauchemin Manon , Garron Marie-Line , Shaya David , Sulea Traian , Cygler Miroslaw , Lau Peter C. K.
Published: December 2007

In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (

T
m

) of the enzyme more than 1 to 2°C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of
Xanthomonas campestris
origin (PL

Xc

) produced a 6°C increase in

T
m

and a 23-fold increase in the half-life at 45°C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established

T
m

values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved

T
m

value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PL

Xc

were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.
The Genetically Remote Pathogenic Strain NVH391-98 of the
Bacillus cereus
Group Is Representative of a Cluster of Thermophilic Strains
Authors: Auger Sandrine , Galleron Nathalie , Bidnenko Elena , Ehrlich S. Dusko , Lapidus Alla , Sorokin Alexei
Published: December 2007

Bacteria of the
Bacillus cereus
group are known to cause food poisoning. A rare phylogenetically remote strain, NVH391-98, was recently characterized to encode a particularly efficient cytotoxin K presumably responsible for food poisoning. This pathogenic strain and its close relatives can be phenotypically distinguished from other strains of the
B. cereus
group by the inability to grow at temperatures below 17°C and by the ability to grow at temperatures from 48 to 53°C. A temperate phage, phBC391A2, residing in the genome of NVH391-98 allows us to distinguish the three known members of this thermophilic strain cluster.
Microbiological Quality of Bagged Cut Spinach and Lettuce Mixes
Authors: Valentin-Bon Iris , Jacobson Andrew , Monday Steven R. , Feng Peter C. H.
Published: December 2007

Analysis of 100 bagged lettuce and spinach samples showed mean total bacterial counts of 7.0 log
10
CFU/g and a broad range of <4 to 8.3 log
10
CFU/g. Most probable numbers (MPN) of ≥11,000 /g coliforms were found in 55 samples, and generic
Escherichia coli
bacteria were detected in 16 samples, but no
E. coli
count exceeded 10 MPN/g.
Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in
Ralstonia eutropha
H16
Authors: Uchino Keiichi , Saito Terumi , Jendrossek Dieter
Published: December 2007

The recently finished genome sequence of
Ralstonia eutropha
H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes (
phaZa1
,
phaZb
,
phaZc
, and
phaZd1
) representing the four classes and investigated the physiological function of the gene products (i) with recombinant
Escherichia coli
strains and (ii) with
R. eutropha
null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.
Genetic Tools for Select-Agent-Compliant Manipulation of
Burkholderia pseudomallei
Authors: Choi Kyoung-Hee , Mima Takehiko , Casart Yveth , Rholl Drew , Kumar Ayush , Beacham Ifor R. , Schweizer Herbert P.
Published: December 2007

Because of
Burkholderia pseudomallei
's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated

Escherichia coli P
BAD

promoter and kanamycin (
nptI
) and zeocin (
ble
) selection markers from the constitutive
Burkholderia thailandensis
ribosomal
P
S12
or synthetic bacterial
P
EM7
promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of
nptII
and
ble
selection markers contained on
FRT
or
loxP
cassettes. Finally, expression of Tn
7
site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for
B. pseudomallei
. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked Δ(
amrRAB
-
oprA
) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different
B. pseudomallei
strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn
7
element carrying the
amrAB
-
oprA
operon. The new tools allow routine select-agent-compliant genetic manipulations of
B. pseudomallei
and other
Burkholderia
species.
Melanin-Based High-Throughput Screen for
l
-Tyrosine Production in
Escherichia coli
Authors: Santos Christine Nicole S. , Stephanopoulos Gregory
Published: December 2007

We present the development of a simple, high-throughput screen for identifying bacterial strains capable of
l
-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link
l
-tyrosine production in
Escherichia coli
with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular
l
-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for
l
-tyrosine-overproducing strains.