bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Fibronectin Type 3-Like Repeat from the
Clostridium thermocellum
Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface
Authors: Kataeva Irina A. , Seidel Ronald D. , Shah Ashit , West Larry T. , Li Xin-Liang , Ljungdahl Lars G. 
Published: August 2002

ABSTRACT

Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of
Clostridium thermocellum
are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn3
1,2
), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn3
1,2
(more efficient), and Gh9-Fn3
1,2
-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn3
1,2
, and then Gh9-Fn3
1,2
-CBDIII (least stable). Mixing of
Orpinomyces
endoglucanase CelE with Fn3
1,2,
or Fn3
1,2
-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn3
1,2
, Fn3
1,2
-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn3
1,2
and Fn3
1,2
-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 μmoles g
−1
and relative affinities (
K
r
) of 1.12 and 2.13 liters g
−1
, respectively. Fn3
1,2
bound weakly to both celluloses. Fn3
1,2
-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 μmoles g
−1
and
K
r
s of 1.14 and 1.98 liters g
−1
, respectively. Fn3
1,2
and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn3
1,2
aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn3
1,2
on the cellulose surface.
Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA
Authors: Gillespie Doreen E. , Brady Sean F. , Bettermann Alan D. , Cianciotto Nicholas P. , Liles Mark R. , Rondon Michelle R. , Clardy Jon , Goodman Robert M. , Handelsman Jo 
Published: August 2002

ABSTRACT

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in
Escherichia coli
with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on
E. coli
; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes,
lly
, which is required for production of the hemolytic pigment in
Legionella pneumophila
, also conferred production of turbomycin A and B on
E. coli
. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by
E. coli
, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.
Seasonal Variation in Lysogeny as Depicted by Prophage Induction in Tampa Bay, Florida
Authors: Williamson S. J. , Houchin L. A. , McDaniel L. , Paul J. H. 
Published: August 2002

ABSTRACT

A seasonal study of the distribution of lysogenic bacteria in Tampa Bay, Florida, was conducted over a 13-month period. Biweekly water samples were collected and either were left unaltered or had the viral population reduced by filtration (pore size, 0.2 μm) and resuspension in filtered (pore size, 0.2 μm) water. Virus-reduced and unaltered samples were then treated by adding mitomycin C (0.5 μg ml
−1
) to induce prophage or were left untreated. In order to test the hypothesis that prophage induction was phosphate limited, additional induction experiments were performed in the presence and absence of phosphate. Induction was assessed as an increase in viral direct counts, relative to those obtained in controls, as detected by epifluorescence microscopy. Induction of prophage was observed in 5 of 25 (20%) unaltered samples which were obtained during or after the month of February, paralleling the results from a previous seasonal study. Induction of prophage was observed in 9 of 25 (36%) of the virus-reduced samples, primarily those obtained in the winter months, which was not observed in a prior seasonal study (P. K. Cochran and J. H. Paul, Appl. Environ. Microbiol. 64:2308-2312, 1998). Induction was noted in the months of lowest bacterial and primary production, suggesting that lysogeny was favored under conditions of poor host growth. Phosphate addition enabled prophage induction in two of nine (22%) experiments. These results indicate that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration, suggesting that phosphate concentration may modulate the lysogenic response of natural populations.
Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of
Ralstonia eutropha
H16
Authors: Overhage Jörg , Steinbüchel Alexander , Priefert Horst 
Published: August 2002

ABSTRACT

The gene loci
ehyAB
,
calA
, and
calB
, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in
Pseudomonas
sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2
ehyABcalAcalB)
and transferred to
Ralstonia eutropha
H16. A recombinant strain of
R. eutropha
H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of
R. eutropha
H16(pBBR1-JO2
ehyABcalAcalB)
from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.
Lactobacillus plantarum
MiLAB 393 Produces the Antifungal Cyclic Dipeptides Cyclo(
l
-Phe-
l
-Pro) and Cyclo(
l
-Phe-
trans
-4-OH-
l
-Pro) and 3-Phenyllactic Acid
Authors: Ström Katrin , Sjögren Jörgen , Broberg Anders , Schnürer Johan 
Published: August 2002

ABSTRACT

We have isolated a
Lactobacillus plantarum
strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay.
Fusarium sporotrichioides
and
Aspergillus fumigatus
were the most sensitive among the molds, and
Kluyveromyces marxianus
was the most sensitive yeast species. No inhibitory activity could be detected against the mold
Penicillium roqueforti
or the yeast
Zygosaccharomyces bailii
. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C
18
SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C
18
column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(
l
-Phe-
l
-Pro), cyclo(
l
-Phe-
trans
-4-OH-
l
-Pro) and 3-phenyllactic acid (
l
/
d
isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against
A. fumigatus
and
P. roqueforti
were 20 mg ml
−1
for cyclo(
l
-Phe-
l
-Pro) and 7.5 mg ml
−1
for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(
l
-Phe-
l
-Pro) and cyclo(
l
-Phe-
trans
-4-OH-
l
-Pro) by lactic acid bacteria is reported here for the first time.
Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of
Pseudomonas putida
KT2440
Authors: Manzanera M. , García de Castro A. , Tøndervik A. , Rayner-Brandes M. , Strøm A. R. , Tunnacliffe A. 
Published: August 2002

ABSTRACT

Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium
Pseudomonas putida
KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in
P. putida
KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For
P. putida
KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.
Role of
tfxE
, but Not
tfxG
, in Trifolitoxin Resistance
Authors: Scupham Alexandra J. , Dong Yuemei , Triplett Eric W. 
Published: August 2002

ABSTRACT

Eight genes,
tfxABCDEFG
and
tfuA
, confer production of trifolitoxin (TFX), a ribosomally synthesized, posttranslationally modified peptide antibiotic, in TFX-sensitive α-proteobacteria. An in-frame deletion in
tfxE
significantly reduced a strain's resistance to TFX in comparison to that of an otherwise identical construct containing wild-type
tfxE
. The deletion of
tfxG
had no effect on TFX resistance. Nevertheless, RNase protection assays showed that
tfxE
and
tfxG
are transcribed, showing that the
tfxDEFG
mRNA was produced on the same transcript. Examination of the role of
tfxG
in TFX production showed that the
tfxG
mutant expressed slightly less TFX activity and produced only one TFX isomer while four are produced by the wild-type strain. Thus,
tfxE
plays an important role in TFX resistance while
tfxG
is important in optimal TFX production through the production of TFX isomers.
Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by
Sphingomonas xenophaga
BN6
Authors: Keck Andreas , Rau Jörg , Reemtsma Thorsten , Mattes Ralf , Stolz Andreas , Klein Joachim 
Published: August 2002

ABSTRACT

During aerobic degradation of naphthalene-2-sulfonate (2NS),
Sphingomonas xenophaga
strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (
nsaC
) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of
S. xenophaga
BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.
Formation and Conversion of Oxygen Metabolites by
Lactococcus lactis
subsp.
lactis
ATCC 19435 under Different Growth Conditions
Authors: van Niel Ed W. J. , Hofvendahl Karin , Hahn-Hägerdal Bärbel 
Published: August 2002

ABSTRACT

A semidefined medium based on Casamino Acids allowed
Lactococcus lactis
ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h
−1
). Accumulation of H
2
O
2
in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H
2
O
2
accumulated only temporarily during the lag phase. H
2
O
2
is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min
−1
mg of protein
−1
), which reduced oxygen to water, whereby superoxide was formed as a by-product. H
2
O
2
originated from the dismutation of superoxide by superoxide dismutase. Although H
2
O
2
was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H
2
O
2
-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H
2
O
2
and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect
L. lactis
strain ATCC 19435 from H
2
O
2
.
Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of
Lactococcus lactis
subsp.
cremoris
Authors: Malone A. S. , Shellhammer T. H. , Courtney P. D. 
Published: August 2002

ABSTRACT

Viability, morphology, lysis, and cell wall hydrolase activity of
Lactococcus lactis
subsp.
cremoris
MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in
L. lactis
. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.
Effect of Exopolysaccharides on Phage-Host Interactions in
Lactococcus lactis
Authors: Deveau Hélène , Van Calsteren Marie-Rose , Moineau Sylvain 
Published: August 2002

ABSTRACT

In this study, we report that
Lactococcus lactis
strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps
+
strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps
−
L. lactis
strains. The host range of these phages was determined with 19
Lactococcus
strains, including 7 Eps
+
and 12 Eps
−
cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps
+
strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps
+
strain SMQ-420. The five other Eps
+
strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps
−
strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps
+
strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the
eps
operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype.
Influence of Elevated CO2on the Fungal Community in a Coastal Scrub Oak Forest Soil Investigated with Terminal-Restriction Fragment Length Polymorphism Analysis
Authors: Klamer Morten , Roberts Michael S. , Levine Lanfang H. , Drake Bert G. , Garland Jay L. 
Published: August 2002

ABSTRACTSixteen open-top chambers (diameter, 3.66 m) were established in a scrub oak habitat in central Florida where vegetation was removed in a planned burn prior to chamber installation. Eight control chambers have been continuously exposed to ambient air and eight have been continuously exposed to elevated CO2at twice-ambient concentration (∼700 ppm) for 5 years. Soil cores were collected from each chamber to examine the influence of elevated atmospheric CO2on the fungal community in different soil fractions. Each soil sample was physically fractionated into bulk soil, rhizosphere soil, and roots for separate analyses. Changes in relative fungal biomass were estimated by the ergosterol technique. In the bulk soil and root fractions, a significantly increased level of ergosterol was detected in the elevated CO2treatments relative to ambient controls. Fungal community composition was determined by terminal-restriction fragment length polymorphism (T-RFLP) analysis of the internal transcribed spacer (ITS) region. The specificities of different ITS primer sets were evaluated against plant and fungal species isolated from the experimental site. Changes in community composition were assessed by principal component analyses of T-RFLP profiles resolved by capillary electrophoresis. Fungal species richness, defined by the total number of terminal restriction fragments, was not significantly affected by either CO2treatment or soil fraction.
Influence of Tropolone on
Poria placenta
Wood Degradation
Authors: Diouf P. N. , Delbarre N. , Perrin D. , Gérardin P. , Rapin C. , Jacquot J. P. , Gelhaye E. 
Published: August 2002

ABSTRACT

Fenton reactions are believed to play important roles in wood degradation by brown rot fungi. In this context, the effect of tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator, on wood degradation by
Poria placenta
was investigated. Tropolone (50 μM) strongly inhibits fungal growth on malt agar, but this inhibition could be relieved by adding iron salts. With an experimental system containing two separate parts, one supplemented with tropolone (100 μM) and the other not, it was shown that the fungus is able to reallocate essential minerals from the area where they are available and also to grow in these conditions on malt-agar in the presence of tropolone. Nevertheless, even in the presence of an external source of metals,
P. placenta
is not able to attack pine blocks impregnated with tropolone (5 mM). This wood degradation inhibition is related to the presence of the tropolone hydroxyl group, as shown by the use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone). Furthermore, tropolone possesses both weak antioxidative and weak radical-scavenging properties and a strong affinity for ferric ion and is able to inhibit ferric iron reduction by catecholates, lowering the redox potential of the iron couple. These data are consistent with the hypothesis that tropolone inhibits wood degradation by
P. placenta
by chelating iron present in wood, thus avoiding initiation of the Fenton reaction. This study demonstrates that iron chelators such as tropolone could be also involved in novel and more environmentally benign preservative systems.