bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin fromLactobacillus plantarumNC8
Authors: Maldonado Antonio , Ruiz-Barba José Luis , Jiménez-Díaz Rufino 
Published: January 2003

ABSTRACTA new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated fromLactobacillus plantarumNC8 cultures which had been induced withLactococcus lactisMG1363 orPediococcus pentosaceusFBB63. This bacteriocin consists of two distinct peptides, named α and β, which were separated by C2-C18reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8α and PLNC8β, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (α) and 4,000 Da (β). The corresponding genes, designatedplNC8AandplNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature α and β peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream ofplNC8Band overlappingplNC8A. Upstream of the putative −35 region ofplNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.
Morphological and Phylogenetic Characterizations of FreshwaterThioplocaSpecies from Lake Biwa, Japan, and Lake Constance, Germany
Authors: Kojima Hisaya , Teske Andreas , Fukui Manabu 
Published: January 2003

ABSTRACTFilamentous, gliding, sulfide-oxidizing bacteria of the genusThioplocawere found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake BiwaThioplocaresembled morphologicallyThioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 μm; the diameters of completeThioplocafilaments ranged from 18 to 75 μm. The cell lengths ranged from 1.2 to 3.8 μm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of theThioplocafrom Lake Biwa was amplified by using newly designedThioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, theThioplocafrom Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake BiwaThioploca. Positive signals were obtained with the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake BiwaThioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, theThioplocafrom Lake Biwa and theThioplocafrom Lake Constance are closely related toT. ingricaand to each other.
Production of Optically Pure
d
-Lactic Acid in Mineral Salts Medium by Metabolically Engineered
Escherichia coli
W3110
Authors: Zhou Shengde , Causey T. B. , Hasona A. , Shanmugam K. T. , Ingram L. O. 
Published: January 2003

ABSTRACT

The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of
l
-lactic acid to
d
-lactic acid. Although the largest demand is for the
l
enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of
Escherichia coli
W3110 (prototrophic) as new biocatalysts for the production of
d
-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction.
d
-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this
d
-lactic acid was ∼98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (
frdABCD
), alcohol/aldehyde dehydrogenase (
adhE
), and pyruvate formate lyase (
pflB
). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (
ackA
). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of
d
-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.
Potential Rates of Fermentation in Digesta from the Gastrointestinal Tract of Pigs: Effect of Feeding Fermented Liquid Feed
Authors: Højberg Ole , Canibe Nuria , Knudsen Bettina , Jensen Bent Borg 
Published: January 2003

ABSTRACT

Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system. The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota. In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum,
P
< 0.1; colon,
P
< 0.01; and rectum,
P
< 0.0001) of pigs fed FLF compared to pigs fed dry feed. No effect of diet was observed in the stomach (
P
= 0.71) or the distal part of the small intestine (
P
= 0.97). The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose. Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids. The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals. This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population. On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of
Escherichia coli
and
Salmonella
spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH. The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization. However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract. The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota.
Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci
Authors: Lunde Merete , Blatny Janet Martha , Lillehaug Dag , Aastveit Are Halvor , Nes Ingolf F. 
Published: January 2003

ABSTRACT

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage φLC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six φLC3 lysogenic
Lactococcus lactis
subsp.
cremoris
host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the φLC3 prophage was found to vary with the growth phase of its host
L. lactis
IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the φLC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.
Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase
Authors: Kamerbeek Nanne M. , Olsthoorn Arjen J. J. , Fraaije Marco W. , Janssen Dick B. 
Published: January 2003

ABSTRACT

The 4-hydroxyacetophenone monooxygenase (HAPMO) from
Pseudomonas fluorescens
ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant
Escherichia coli
, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1
R
,5
S
)-bicyclohept-2-en-6-one, with an enantiomeric ratio (
E
) of 20, thus enabling kinetic resolution to obtain the (1
S
,5
R
) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (
ee
) for the two lactones obtained [77%
ee
for (1
S
,5
R
)-2 and 34%
ee
for (1
R
,5
S
)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (
S
)-sulfoxides (
ee
> 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.
2-Hydroxypenta-2,4-dienoate Metabolic Pathway Genes in a Strong Polychlorinated Biphenyl Degrader,
Rhodococcus
sp. Strain RHA1
Authors: Sakai Masayuki , Miyauchi Keisuke , Kato Noboru , Masai Eiji , Fukuda Masao 
Published: January 2003

ABSTRACT

A gram-positive polychlorinated biphenyl (PCB) degrader,
Rhodococcus
sp. strain RHA1, metabolizes biphenyl through the 2-hydroxypenta-2,4-dienoate (HPD) and benzoate metabolic pathways. The HPD metabolic pathway genes, the HPD hydratase (
bphE1
), 4-hydroxy-2-oxovalerate aldolase (
bphF1
), and acetaldehyde dehydrogenase (acylating) (
bphG
) genes, were cloned from RHA1. The deduced amino acid sequences of
bphGF1E1
have 30 to 58% identity with those of the HPD metabolic pathway genes of gram-negative bacteria. The order of these genes,
bphG
-
bphF1
-
bphE1
, differs from that of the HPD metabolic pathway genes,
bphE
-
bphG
-
bphF
, in gram-negative degraders of PCB, phenol, and toluene. Reverse transcription-PCR experiments indicated that the
bphGF1E1
genes are inducibly cotranscribed in cells grown on biphenyl and ethylbenzene. Primer extension analysis revealed that the transcriptional initiation site exists within the
bphR
gene located adjacent to and upstream of
bphG
, which is deduced to code a transcriptional regulator. The respective enzyme activities of
bphGF1E1
gene products were detected in
Rhodococcus erythropolis
IAM1399 carrying a
bphGF1E1
plasmid. The insertional inactivation of the
bphE1
,
bphF1
, and
bphG
genes resulted in the loss of the corresponding enzyme activities and diminished growth on both biphenyl and ethylbenzene. Severe growth interference was observed during growth on biphenyl. The growth defects were partially restored by the introduction of plasmids containing the respective intact genes. These results indicated that the cloned
bphGF1E1
genes are not only responsible for the primary metabolism of HPD during growth on both biphenyl and ethylbenzene but are also involved in preventing the accumulation of unexpected toxic metabolites, which interfere with the growth of RHA1.
Biodegradation of an Alicyclic Hydrocarbon by a Sulfate-Reducing Enrichment from a Gas Condensate-Contaminated Aquifer
Authors: Rios-Hernandez Luis A. , Gieg Lisa M. , Suflita Joseph M. 
Published: January 2003

ABSTRACT

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera
Syntrophobacter
and
Desulfotomaculum
as well as a member of the
Cytophaga-Flexibacter-Bacteroides
group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.
Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis
Authors: Ennahar Saïd , Cai Yimin , Fujita Yasuhito 
Published: January 2003

ABSTRACT

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera
Enterococcus
,
Lactobacillus
,
Lactococcus
,
Leuconostoc
,
Pediococcus
, and
Weissella
. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of
Lactobacillus plantarum
(24%),
Lactococcus lactis
(22%),
Leuconostoc pseudomesenteroides
(20%),
Pediococcus acidilactici
(11%),
Lactobacillus brevis
(11%),
Enterococcus faecalis
(7%),
Weissella kimchii
(3%), and
Pediococcus pentosaceus
(2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.
Compost-Induced Suppression of
Pythium
Damping-Off Is Mediated by Fatty-Acid-Metabolizing Seed-Colonizing Microbial Communities
Authors: McKellar Mary E. , Nelson Eric B. 
Published: January 2003

ABSTRACT

Leaf composts were studied for their suppressive effects on
Pythium ultimum
sporangium germination, cottonseed colonization, and the severity of
Pythium
damping-off of cotton. A focus of the work was to assess the role of fatty-acid-metabolizing microbial communities in disease suppression. Suppressiveness was expressed within the first few hours of seed germination as revealed by reduced
P
.
ultimum
sporangium germination, reduced seed colonization, and reduced damping-off in transplant experiments. These reductions were not observed when cottonseeds were sown in a conducive leaf compost. Microbial consortia recovered from the surface of cottonseeds during the first few hours of germination in suppressive compost (suppressive consortia) induced significant levels of damping-off suppression, whereas no suppression was induced by microbial consortia recovered from cottonseeds germinated in conducive compost (conducive consortia). Suppressive consortia rapidly metabolized linoleic acid, whereas conducive consortia did not. Furthermore, populations of fatty-acid-metabolizing bacteria and actinobacteria were higher in suppressive consortia than in conducive consortia. Individual bacterial isolates varied in their ability to metabolize linoleic acid and protect seedlings from damping-off. Results indicate that communities of compost-inhabiting microorganisms colonizing cottonseeds within the first few hours after sowing in a
Pythium
-suppressive compost play a major role in the suppression of
P
.
ultimum
sporangium germination, seed colonization, and damping-off. Results further indicate that fatty acid metabolism by these seed-colonizing bacterial consortia can explain the
Pythium
suppression observed.
In Situ Exposure to Low Herbicide Concentrations Affects Microbial Population Composition and Catabolic Gene Frequency in an Aerobic Shallow Aquifer
Authors: de Lipthay Julia R. , Tuxen Nina , Johnsen Kaare , Hansen Lars H. , Albrechtsen Hans-Jørgen , Bjerg Poul L. , Aamand Jens 
Published: January 2003

ABSTRACT

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 μg l
−1
) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations,
Pseudomonas
bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10
0
to 10
4
g
−1
sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes
tfdA
and
tfdB
(10
2
to 10
3
gene copies g
−1
sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of
tfd
genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of
Pseudomonas
bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4′,6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.
Online Monitoring of
Escherichia coli
Ghost Production
Authors: Haidinger W. , Szostak M. P. , Jechlinger W. , Lubitz W. 
Published: January 2003

ABSTRACT

Controlled expression of cloned φX174 gene
E
in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.