bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of a Novel Glucosyltransferase from
Lactobacillus reuteri
Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds
Authors: Kralj S. , van Geel-Schutten G. H. , Rahaoui H. , Leer R. J. , Faber E. J. , van der Maarel M. J. E. C. , Dijkhuizen L.
Published: August 2002

Lactobacillus reuteri
strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (
gtf
) genes from lactic acid bacteria, the
L. reuteri
strain 121 glucosyltransferase gene (
gtfA
) was isolated. The
gtfA
open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced
M
r
of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The
gtfA
gene was expressed in
Escherichia coli
, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the
L. reuteri
strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the
gtfA
ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of
L. reuteri
strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in
L. reuteri
strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a
Lactobacillus
strain.
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

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

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

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

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

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

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

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

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

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

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

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

Sixteen 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

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.
Assessment of the Importance of Similarity in Carbon Source Utilization Profiles between the Biological Control Agent and the Pathogen in Biological Control of Bacterial Speck of Tomato
Authors: Ji Pingsheng , Wilson Mark
Published: August 2002

Bacterial speck of tomato, caused byPseudomonas syringaepv. tomato, was used to determine whether similarity in carbon source utilization between a preemptive biological control agent and the pathogen was significant in determining the ability of the bacterium to suppress disease. Similarity in carbon source utilization was quantified as the ratio of the number of tomato carbon sources utilized in vitro by the biological control agent to the number of tomato carbon sources utilized in vitro by the target pathogen (the niche overlap index [NOI]). Suppression of the disease was quantified as the percent reduction in disease severity compared to the pathogen-only control when nonpathogenic bacteria were applied to foliage 48 h prior to the pathogen. In the collection of 36 nonpathogenic bacterial strains, there was a significant (P< 0.01), but weak (r2= 0.25), correlation between reduction in disease severity and similarity in carbon source utilization, suggesting that similarity in carbon source use was significant in determining ability to suppress disease. The relationship was investigated further using catabolic mutants ofP. syringaestrain TLP2, an effective biological control agent of speck. Catabolic mutants exhibited lower levels of similarity (NOI = 0.07 to 0.90) than did wild-type TLP2 (NOI = 0.93). With these catabolic mutants there was a significant (P< 0.01), and stronger (r2= 0.42), correlation between reduction in disease severity and similarity in carbon source utilization. This suggests that similarity in carbon source utilization was a more important component of biological control ability for the catabolic mutants than for the nonpathogenic bacteria. Together, these studies indicate that suppression of bacterial speck of tomato was correlated with nutritional similarity between the pathogenic and nonpathogenic bacteria and suggest that preemptive utilization of carbon sources was probably involved in the biological control of the disease by both the naturally occurring nonpathogenic bacteria and the catabolic mutants.
Characterization of a Novel Fructosyltransferase from
Lactobacillus reuteri
That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides
Authors: van Hijum S. A. F. T. , van Geel-Schutten G. H. , Rahaoui H. , van der Maarel M. J. E. C. , Dijkhuizen L.
Published: August 2002

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from
Lactobacillus reuteri
strain 121. A C-terminally truncated version of the
ftf
gene was successfully expressed in
Escherichia coli
. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2
→
1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10
7
) with β-(2
→
1) linkages (an inulin). FOS, but not inulin, was found in supernatants of
L. reuteri
strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only
Streptococcus mutans
strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a
Lactobacillus ftf
gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an
L. reuteri
strain remain to be established.
In Situ Determination of the Intracellular pH ofLactococcus lactisandLactobacillus plantarumduring Pressure Treatment
Authors: Molina-Gutierrez Adriana , Stippl Volker , Delgado Antonio , Gänzle Michael G. , Vogel Rudi F.
Published: August 2002

Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs ofLactococcus lactisandLactobacillus plantarumduring and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts ofLactococcus lactisandLactobacillus plantarumby 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH ofL. lactiswas decreased to the value of the extracellular pH during compression. The same result was observed during treatment ofLactobacillus plantarumat 300 MPa.Lactobacillus plantarumwas unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5.Lactococcus lactislost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions.
Characterization of the 5-Carboxyvanillate Decarboxylase Gene and Its Role in Lignin-Related Biphenyl Catabolism in
Sphingomonas paucimobilis
SYK-6
Authors: Peng Xue , Masai Eiji , Kitayama Hirotaka , Harada Kyo , Katayama Yoshihiro , Fukuda Masao
Published: August 2002

Sphingomonas paucimobilis
SYK-6 degrades a lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA
O
-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the
meta
-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate,
O
-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb
Eco
RI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it
ligW
. The gene product of
ligW
(LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented
meta
to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the
ligW
gene was located on the 18-kb DNA region with other DDVA catabolic genes, including
ligZ
,
ligY
, and
ligX
.
Cloning and Characterization of the Ferulic Acid Catabolic Genes of
Sphingomonas paucimobilis
SYK-6
Authors: Masai Eiji , Harada Kyo , Peng Xue , Kitayama Hirotaka , Katayama Yoshihiro , Fukuda Masao
Published: August 2002

Sphingomonas paucimobilis
SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn
5
mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb
Bam
HI-
Eco
RI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on
Escherichia coli
host cells. A sequencing analysis revealed the genes
ferB
and
ferA
in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of
ferB
showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of
Pseudomonas
and
Amycolatopsis
ferulic acid degraders. On the other hand, the deduced amino acid sequence of
ferA
showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of
ferA
did show 31% identity with pimeloyl-CoA synthetase of
Pseudomonas mendocina
35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of
E. coli
carrying each of these genes,
ferA
and
ferB
were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively.
p
-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a
ferB
homolog,
ferB2
, upstream of a 5-carboxyvanillic acid decarboxylase gene (
ligW
) involved in the degradation of 5,5′-dehydrodivanillic acid. The deduced amino acid sequence of
ferB2
showed 49% identity with
ferB
, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB. Insertional inactivation of each
fer
gene in
S. paucimobilis
SYK-6 suggested that the
ferA
gene is essential and that
ferB
and
ferB2
genes are involved in ferulic acid degradation.
Environmental Factors That Control Microbial Perchlorate Reduction
Authors: Chaudhuri Swades K. , O'Connor Susan M. , Gustavson Ruth L. , Achenbach Laurie A. , Coates John D.
Published: August 2002

As part of a study to elucidate the environmental parameters that control microbial perchlorate respiration, we investigated the reduction of perchlorate by the dissimilatory perchlorate reducer
Dechlorosoma suillum
under a diverse set of environmental conditions. Our results demonstrated that perchlorate reduction by
D. suillum
only occurred under anaerobic conditions in the presence of perchlorate and was dependent on the presence of molybdenum. Perchlorate reduction was dependent on the presence of the enzyme chlorite dismutase, which was induced during metabolism of perchlorate. Anaerobic conditions alone were not enough to induce expression of this enzyme. Dissolved oxygen concentrations less than 2 mg liter
−1
were enough to inhibit perchlorate reduction by
D. suillum
. Similarly to oxygen, nitrate also regulated chlorite dismutase expression and repressed perchlorate reduction by
D. suillum.
Perchlorate-grown cultures of
D. suillum
preferentially reduced nitrate in media with equimolar amounts of perchlorate and nitrate. In contrast, an extended (40 h) lag phase was observed if a similar nitrate-perchlorate medium was inoculated with a nitrate-grown culture. Perchlorate reduction commenced only when nitrate was completely removed in either of these experiments. In contrast to
D. suillum
, nitrate had no inhibitory effects on perchlorate reduction by the perchlorate reducer
Dechloromonas agitata
strain CKB. Nitrate was reduced to nitrite concomitant with perchlorate reduction to chloride. These studies demonstrate that microbial respiration of perchlorate is significantly affected by environmental conditions and perchlorate reduction is directly dependent on bioavailable molybdenum and the presence or absence of competing electron acceptors. A microbial treatment strategy can achieve and maintain perchlorate concentrations below the recommended regulatory level, but only in environments in which the variables described above can be controlled.
Molecular Evidence for a Uniform Microbial Community in Sponges from Different Oceans
Authors: Hentschel Ute , Hopke Jörn , Horn Matthias , Friedrich Anja B. , Wagner Michael , Hacker Jörg , Moore Bradley S.
Published: August 2002

Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges
Aplysina aerophoba
and
Theonella swinhoei
were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.
Effect of Modified Atmosphere Composition on the Metabolism of Glucose byBrochothrix thermosphacta
Authors: Pin Carmen , García de Fernando Gonzalo D. , Ordóñez Juan A.
Published: August 2002

The influence of atmosphere composition on the metabolism ofBrochothrix thermosphactawas studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose byB. thermosphactawas influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO2percentages, glucose metabolism remained anaerobic under greater oxygen contents.
Metabolic Control Analysis of Glycerol Synthesis inSaccharomyces cerevisiae
Authors: Cronwright Garth R. , Rohwer Johann M. , Prior Bernard A.
Published: August 2002

Glycerol, a major by-product of ethanol fermentation bySaccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux inS. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD+-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (Cv1J) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (RDHAPJ= 0.48 to 0.69), followed by ATP concentration (RATPJ= −0.21 to −0.50). Interestingly, the pathway responds weakly to NADH concentration (RNADHJ= 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.
Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of
Pseudomonas aeruginosa
Biofilms
Authors: Purevdorj B. , Costerton J. W. , Stoodley P.
Published: August 2002

Biofilms were grown from wild-type (WT)
Pseudomonas aeruginosa
PAO1 and the cell signaling
lasI
mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (
P
< 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.
Optimization of Bacteriocin Production by Batch Fermentation of
Lactobacillus plantarum
LPCO10
Authors: Leal-Sánchez M. V. , Jiménez-Díaz R. , Maldonado-Barragán A. , Garrido-Fernández A. , Ruiz-Barba J. L.
Published: August 2002

Optimization of bacteriocin production by
Lactobacillus plantarum
LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2
3
5-2
fractional factorial two-level design and tested for their influence on maximal bacteriocin production by
L. plantarum
LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3
2
3-1
fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and
L. plantarum
LPCO10 inoculum size ranging from 10
7.3
to 10
7.4
CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10
4
times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from
L. plantarum
LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from
L. plantarum
LPCO10 so that bacteriocins can be used as preservatives in canned foods.
Molecular Evolution of Aromatic Polyketides and Comparative Sequence Analysis of Polyketide Ketosynthase and 16S Ribosomal DNA Genes from Various
Streptomyces
Species
Authors: Metsä-Ketelä Mikko , Halo Laura , Munukka Eveliina , Hakala Juha , Mäntsälä Pekka , Ylihonko Kristiina
Published: August 2002

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the γ-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.
Soil-Atmosphere CO Exchanges and Microbial Biogeochemistry of CO Transformations in a Brazilian Agricultural Ecosystem
Authors: King Gary M. , Hungria M.
Published: August 2002

Although anthropogenic land use has major impacts on the exchange of soil and atmosphere gas in general, relatively little is known about its impacts on carbon monoxide. We compared soil-atmosphere CO exchanges as a function of land use, crop type, and tillage treatment on an experimental farm in Parãna, Brazil, that is representative of regionally important agricultural ecosystems. Our results showed that cultivated soils consumed CO at rates between 3 and 6 mg of CO m
−2
day
−1
, with no statistically significant effect of tillage method or crop. However, CO exchange for a pasture soil was near zero, and an unmanaged woodlot emitted CO at a rate of 9 mg of CO m
−2
day
−1
. Neither nitrite, aluminum sulfate, nor methyl fluoride additions affected CO consumption by tilled or untilled soils from soybean plots, indicating that CO oxidation did not depend on ammonia oxidizers and that CO oxidation patterns differed in part from patterns reported for forest soils. The apparent

K
m

for CO uptake, 5 to 11 ppm, was similar to values reported for temperate forest soils;
V
max
values, approximately 1 μg of CO g (dry weight)
−1
h
−1
, were comparable for woodlot and cultivated soils in spite of the fact that the latter consumed CO under ambient conditions. Short-term (24-h) exposure to elevated levels of CO (10% CO) partially inhibited uptake at lower concentrations (i.e., 100 ppm), suggesting that the sensitivity to CO of microbial populations that are active in situ differs from that of known carboxydotrophs. Soil-free soybean and corn roots consumed CO when they were incubated with 100-ppm concentrations and produced CO when they were incubated with ambient concentrations. These results document for the first time a role for cultivated plant roots in the dynamics of CO in an agricultural ecosystem.