bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane
Authors: Alakomi H.-L. , Skyttä E. , Saarela M. , Mattila-Sandholm T. , Latva-Kala K. , Helander I. M. 
Published: July 2002

ABSTRACT

The effect of lactic acid on the outer membrane permeability of
Escherichia coli
O157:H7,
Pseudomonas aeruginosa
, and
Salmonella enterica
serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for
P. aeruginosa
, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl
2
. Lactic acid sensitized
E. coli
and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized
P. aeruginosa
to SDS and to Triton X-100.
P. aeruginosa
was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.
A New
Klebsiella planticola
Strain (Cd-1) Grows Anaerobically at High Cadmium Concentrations and Precipitates Cadmium Sulfide
Authors: Sharma Pramod K. , Balkwill David L. , Frenkel Anatoly , Vairavamurthy Murthy A. 
Published: July 2002

ABSTRACT

Heavy metal resistance by bacteria is a topic of much importance to the bioremediation of contaminated soils and sediments. We report here the isolation of a highly cadmium-resistant
Klebsiella planticola
strain, Cd-1, from reducing salt marsh sediments. The strain grows in up to 15 mM CdCl
2
under a wide range of NaCl concentrations and at acidic or neutral pH. In growth medium amended with thiosulfate, it precipitated significant amounts of cadmium sulfide (CdS), as confirmed by x-absorption spectroscopy. In comparison with various other strains tested, Cd-1 is superior for precipitating CdS in cultures containing thiosulfate. Thus, our results suggest that Cd-1 is a good candidate for the accelerated bioremediation of systems contaminated by high levels of cadmium.
rpoB
-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity
Authors: Dahllöf Ingela , Baillie Harriet , Kjelleberg Staffan 
Published: July 2002

ABSTRACT

Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (
rpoB
), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga
Delisea pulchra
and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of
rpoB
, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and
rpoB
PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from
D. pulchra
. Eight out of 14 isolates from
D. pulchra
, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when
rpoB
was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from
D. pulchra
proved to be more accurately described using
rpoB
, compared to the 16S rDNA-based PCR-DGGE.
Mathematical Analysis of Growth and Interaction Dynamics of Streptomycetes and a Bacteriophage in Soil
Authors: Burroughs N. J. , Marsh P. , Wellington E. M. H. 
Published: July 2002

ABSTRACTWe observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its hostStreptomyces lividansTK24 in sterile soil microcosms. Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts. The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens. The interaction ofS. lividansand KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments. This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics. Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates. There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (>150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean [SEM], 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage. We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil.
Application of the 5′-Nuclease PCR Assay in Evaluation and Development of Methods for Quantitative Detection ofCampylobacter jejuni
Authors: Nogva Hege Karin , Bergh Anette , Holck Askild , Rudi Knut 
Published: July 2002

ABSTRACTCampylobacter jejuniis recognized as a leading human food-borne pathogen. Traditional diagnostic testing forC. jejuniis not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5′-nuclease PCR assay for quantitative detection ofC. jejuniand describe its evaluation. A probe including positions 381121 to 381206 of the publishedC. jejunistrain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates ofC. jejunitested (32 isolates, including the type strain) and negative for all otherCampylobacterspp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development ofC. jejunidetection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viableC. jejuniafter exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from deadC. jejuniand protected DNA in living bacteria.
Detection of EnterotoxicBacillus cereusandBacillus thuringiensisStrains by PCR Analysis
Authors: Hansen Bjarne Munk , Hendriksen Niels Bohse 
Published: July 2002

ABSTRACTMany strains ofBacillus cereuscause gastrointestinal diseases, and the closely related insect pathogenB. thuringiensishas also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22B. cereusand 41B. thuringiensisstrains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of theB. thuringiensisstrains, while sixB. cereusstrains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate thatbceTis widely distributed amongB. cereusandB. thuringiensisstrains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected thebceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceTgene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereusandB. thuringiensisbased on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection ofbceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.
Pantoea agglomeransStrain EH318 Produces Two Antibiotics That InhibitErwinia amylovoraIn Vitro
Authors: Wright Sandra A. I. , Zumoff Cathy H. , Schneider Lois , Beer Steven V. 
Published: July 2002

ABSTRACTPantoea agglomerans(synonym:Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded withErwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred onEscherichia colithe ability to inhibit growth ofE. amylovora. The two cosmids conferred different antibiotic activities onE. coliDH5α and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-succinamic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.
Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA
Authors: Demanèche Sandrine , Jocteur-Monrozier Lucile , Quiquampoix Hervé , Simonet Pascal 
Published: July 2002

ABSTRACT

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium
Acinetobacter
sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.
Genetic Engineering of
Escherichia coli
for Enhanced Uptake and Bioaccumulation of Mercury
Authors: Bae Weon , Mehra Rajesh K. , Mulchandani Ashok , Chen Wilfred 
Published: July 2002

ABSTRACT

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)

n

Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg
2+
transport system with (Glu-Cys)
20
Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg
2+
. Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.
Identification of Bacteria Responsible for Ammonia Oxidation in Freshwater Aquaria
Authors: Burrell Paul C. , Phalen Carol M. , Hovanec Timothy A. 
Published: July 2002

ABSTRACT

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the
Nitrosomonas marina
cluster, the
Nitrosospira
cluster, or the
Nitrosomonas europaea-Nitrosococcus mobilis
cluster of the β subdivision of the class
Proteobacteria
. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the
Nitrosomonas marina
-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the
Nitrosomonas marina
-like AOB strain was present in the original enrichment, even one with other AOB, only the
Nitrosomonas marina
-like AOB strain was present in aquaria after nitrification was established.
Nitrosomonas marina
-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.
Expression of
cmg1
, an Exo-β-1,3-Glucanase Gene from
Coniothyrium minitans
, Increases during Sclerotial Parasitism
Authors: Giczey Gábor , Kerényi Zoltán , Fülöp László , Hornok László 
Published: July 2002

ABSTRACT

During sclerotial infection of
Sclerotinia sclerotiorum
the mycoparasite
Coniothyrium minitans
penetrates through the host cell wall, which contains β-1,3-glucan as its major component. A PCR-based strategy was used to clone a β-1,3-glucanase-encoding gene, designated
cmg1
, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-β-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73.
Saccharomyces cerevisiae
INVSc1 expressing the
cmg1
gene secreted a ∼100-kDa β-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of
S. sclerotiorum
by 35 and 85% at concentrations of 300 and 600 μg ml
−1
, respectively. A single copy of the
cmg1
gene is present in the genome of
C. minitans
. Northern analyses indicated increases in the transcript levels of
cmg1
due to both carbon starvation and the presence of ground sclerotia of
S. sclerotiorum
; only slight repression was observed in the presence of 2% glucose. Expression of
cmg1
increased during parasitic interaction with
S. sclerotiorum
.
TheBacillus thuringiensis cytGenes for Hemolytic Endotoxins Constitute a Gene Family
Authors: Guerchicoff Alejandra , Delécluse Armelle , Rubinstein Clara P. 
Published: July 2002

ABSTRACTIn the same way thatcrygenes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, thecytgenes for hemolytic toxins seem to compose another set of highly related genes inBacillus thuringiensis. Although the occurrence of Cyt hemolytic factors inB. thuringiensishas been typically associated with mosquitocidal strains, we have recently shown thatcytgenes are also present in strains with different pathotypes; this is the case for themorrisonisubspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such asB. thuringiensissubsp.israelensisand subsp.morrisoniPG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50B. thuringiensisstrains revealed thatcyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of manyB. thuringiensisstrains.
Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by
Lactobacillus acidophilus
Authors: Ogawa Jun , Matsumura Kenji , Kishino Shigenobu , Omura Yoriko , Shimizu Sakayu 
Published: July 2002

ABSTRACT

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of
Lactobacillus acidophilus
AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as
cis
-9,
trans
-11- or
trans
-9,
cis
-11-octadecadienoic acid and
trans
-9,
trans
-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-
cis
-12-octadecaenoic acid and 10-hydroxy-
trans
-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-
cis
-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of
L. acidophilus
, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of
L. acidophilus
producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.
Tests of a Cellular Model for Constant Branch Distribution in the Filamentous Fungus
Neurospora crassa
Authors: Watters Michael K. , Griffiths Anthony J. F. 
Published: July 2002

ABSTRACT

The growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. In
Neurospora crassa
, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. Much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by
N. crassa
has not been proposed. We described and tested a model in which the formation of a lateral branch in
N. crassa
was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate of deposition at the apex. If both rates are proportional to metabolic rate, then the model explains the known lack of dependence of branch interval on growth rate. We tested the model by manipulating the tip extension rate, first by shifting temperature in both the wild type and hyperbranching (colonial) mutants and also by observing the behavior of both tipless colonies and colonyless tips. We found that temperature shifts in either direction result in temporary changes in branching. We found that colonyless tips also pass through a temporary transition phase of branching. The tipless colonies produced a cluster of new tips near the point of damage. We also found that branching in colonial mutants is dependent on growth rate. The results of these tests are consistent with a model of branching in which branch initiation is controlled by the dynamics of tip growth while being independent of the actual rate of this growth.