bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diversity and Abundance of Aerobic and Anaerobic Methane Oxidizers at the Haakon Mosby Mud Volcano, Barents Sea
Authors: Lösekann Tina , Knittel Katrin , Nadalig Thierry , Fuchs Bernhard , Niemann Helge , Boetius Antje , Amann Rudolf 
Published: March 2007

ABSTRACT

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with
Methylobacter
and
Methylophaga
species accounted for 56% ± 8% of total cells. In sediments below
Beggiatoa
mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the
Desulfobulbus
(DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94% ± 2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (
Siboglinidae
, formerly known as
Pogonophora
). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and
Beggiatoa
species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (
mcrA
) genes showed a strong dominance of a novel lineage,
mcrA
group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.
Proteomic Approach for Characterization of Hop-Inducible Proteins in
Lactobacillus brevis
Authors: Behr Jürgen , Israel Lars , Gänzle Michael G. , Vogel Rudi F. 
Published: March 2007

ABSTRACT

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-α-acids, are described as ionophores which exchange H
+
for cellular divalent cations, e.g., Mn
2+
, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of
Lactobacillus brevis
TMW 1.465 and hop adaptation in its variant
L. brevis
TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in
L. brevis
TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.
Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by
Propionibacterium freudenreichii
ET-3 Fed-Batch Culture
Authors: Furuichi Keisuke , Katakura Yoshio , Ninomiya Kazuaki , Shioya Suteaki 
Published: March 2007

ABSTRACT

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of
Propionibacterium freudenreichii
ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.
Characterization of Genetically Matched Isolates of
Campylobacter jejuni
Reveals that Mutations in Genes Involved in Flagellar Biosynthesis Alter the Organism's Virulence Potential
Authors: Malik-Kale Preeti , Raphael Brian H. , Parker Craig T. , Joens Lynn A. , Klena John D. , Quiñones Beatriz , Keech Amy M. , Konkel Michael E. 
Published: March 2007

ABSTRACT

Phenotypic and genotypic evidence suggests that not all
Campylobacter jejuni
isolates are pathogenic for humans. We hypothesized that differences in gene content or gene expression alter the degree of pathogenicity of
C. jejuni
isolates. A
C. jejuni
isolate (Turkey) recovered from a turkey and a second
C. jejuni
isolate (CS) recovered from a chicken differed in their degrees of in vitro and in vivo virulence. The
C. jejuni
Turkey isolate invaded INT 407 human epithelial cells and secreted the Cia (
Campylobacter i
nvasion
a
ntigen) proteins, while the
C. jejuni
CS isolate was noninvasive for human epithelial cells and did not secrete the Cia proteins. Newborn piglets inoculated with the
C. jejuni
Turkey isolate developed more severe clinical signs of campylobacteriosis than piglets inoculated with the
C. jejuni
CS isolate. Additional work revealed that flagellin was not expressed in the
C. jejuni
CS isolate. Microarray and real-time reverse transcription-PCR analyses revealed that all flagellar class II genes were significantly downregulated in the
C. jejuni
CS isolate compared to the
C. jejuni
Turkey isolate. Finally, nucleotide sequencing of the
flgR
gene revealed the presence of a single residue that was different in the FlgR proteins of the
C. jejuni
Turkey and CS isolates. Complementation of the
C. jejuni
CS isolate with a wild-type copy of the
flgR
gene restored the isolate's motility. Collectively, these findings support the hypothesis that critical differences in gene content or gene expression can alter the pathogenic potential of
C. jejuni
isolates.
Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments
Authors: Jensen Marlene Mark , Thamdrup Bo , Dalsgaard Tage 
Published: March 2007

ABSTRACTThe effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2production were investigated by using short anoxic incubations of marine sediment with a15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM−1, and there was 95% inhibition at ∼30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM−1; 95% inhibition at ∼70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM−1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ∼50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2production in marine sediments and the use of15N isotope methods for their quantification.
Inhibition of Quorum Sensing in
Pseudomonas aeruginosa
by
N
-Acyl Cyclopentylamides
Authors: Ishida Takenori , Ikeda Tsukasa , Takiguchi Noboru , Kuroda Akio , Ohtake Hisao , Kato Junichi 
Published: March 2007

ABSTRACT

N
-Octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in
Pseudomonas aeruginosa
PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in
P. aeruginosa
PAO1. The
lasB
-
lacZ
and
rhlA
-
lacZ
reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing.
N
-Decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for
lasB
-
lacZ
and
rhlA
-
lacZ
expression were 80 and 90 μM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of
P. aeruginosa
PAO1. C10-CPA inhibited induction of both
lasI
-
lacZ
by
N
-(3-oxododecanoyl)-
l
-homoserine lactone (PAI1) and
rhlA
-
lacZ
by
N
-butanoyl-
l
-homoserine lactone (PAI2) in the
lasI rhlI
mutant of
P. aeruginosa
PAO1, indicating that C10-CPA interferes with the
las
and
rhl
quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.
SYBR Green Real-Time PCR Method To Detect
Clostridium botulinum
Type A
Authors: Fenicia Lucia , Anniballi Fabrizio , De Medici Dario , Delibato Elisabetta , Aureli Paolo 
Published: March 2007

ABSTRACT

Botulinum toxins (BoNTs) are classically produced by
Clostridium botulinum
but rarely also from neurotoxigenic strains of
Clostridium baratii
and
Clostridium butyricum
. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of
C. botulinum
. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting
C. botulinum
type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of
C. botulinum
type A, 4 strains of
C. botulinum
type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 10
1
copies of
C. botulinum
type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of
C. botulinum
types A and Ab (inclusivity and exclusivity, 100%).
Impacts of Cultivation of Marine Diatoms on the Associated Bacterial Community
Authors: Sapp Melanie , Wichels Antje , Gerdts Gunnar 
Published: March 2007

ABSTRACT

The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging to members of the
Gammaproteobacteria
, were observed during cultivation.
Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium
Authors: Schubert Torsten , Maskow Thomas , Benndorf Dirk , Harms Hauke , Breuer Uta 
Published: March 2007

ABSTRACT

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes
ectABC
of the halophilic bacterium
Chromohalobacter salexigens
into an
Escherichia coli
strain using the expression vector pASK-IBA7. Under the control of a
tet
promoter, the transgenic
E. coli
synthesized 6 g liter
−1
ectoine with a space-time yield of 40 mg liter
−1
h
−1
, with the vast majority of the ectoine being excreted.
Prevalence of
Campylobacter
spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine
Campylobacter jejuni
Strains
Authors: Hakkinen Marjaana , Heiska Helmi , Hänninen Marja-Liisa 
Published: March 2007

ABSTRACT

The study investigated the prevalence of
Campylobacter
spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (
n
= 952) and carcass surface samples (
n
= 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples.
Campylobacter jejuni
was isolated from 19.5%,
Campylobacter coli
from 2.2%, and presumptive
Campylobacter hyointestinalis
from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (
n
= 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal
C. jejuni
isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of
C. jejuni
by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass
C. jejuni
isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187
C. jejuni
isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.
Stable Isotope Probing with
15
N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density
Authors: Buckley Daniel H. , Huangyutitham Varisa , Hsu Shi-Fang , Nelson Tyrrell A. 
Published: March 2007

ABSTRACT

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with
15
N, nearly all applications of this technique to date have used
13
C. Successful application of SIP using
15
N-DNA (
15
N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml
−1
for
15
N-labeled DNA, relative to 0.036 g ml
−1
for
13
C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml
−1
. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of
15
N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate
15
N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of
15
N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use
15
N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in
13
C-DNA-SIP applications.
Stable Isotope Probing with
15
N
2
Reveals Novel Noncultivated Diazotrophs in Soil
Authors: Buckley Daniel H. , Huangyutitham Varisa , Hsu Shi-Fang , Nelson Tyrrell A. 
Published: March 2007

ABSTRACT

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of
nifH
gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel
15
N
2
-DNA stable isotope probing (
5
N
2
-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from
15
N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the
Rhizobiales
while the other two represent deeply divergent lineages of noncultivated bacteria within the
Betaproteobacteria
and
Actinobacteria
, respectively. Analysis of
nifH
genes from
15
N-labeled DNA also revealed three microbial groups, one of which was associated with
Alphaproteobacteria
while the others were associated with two noncultivated groups that are deeply divergent within
nifH
cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that
15
N
2
-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by
Actinobacteria
outside of the order
Actinomycetales
.
Characterization of Cultures Enriched from Acidic Polycyclic Aromatic Hydrocarbon-Contaminated Soil for Growth on Pyrene at Low pH
Authors: Uyttebroek Maarten , Vermeir Steven , Wattiau Pierre , Ryngaert Annemie , Springael Dirk 
Published: March 2007

ABSTRACT

Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [
14
C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to
Mycobacterium montefiorense
, belonging to the slow-growing
Mycobacterium
sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by
Pseudomonas putida
and a fast-growing
Mycobacterium
sp. The
M. montefiorense
-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant
Mycobacterium
species in the acidic soils. Our data indicate that a slow-growing
Mycobacterium
species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.
Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by
Thermobifida fusca
Cel9A
Authors: Li Yongchao , Irwin Diana C. , Wilson David B. 
Published: March 2007

ABSTRACT

Thermobifida fusca
Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual
T. fusca
enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in
Escherichia coli
; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.