bluuebunny/crossref_metadata_2025 huggingface dataset:
Ratios of Carbon Isotopes in Microbial Lipids as an Indicator of Substrate Usage
Authors: Abraham Wolf-Rainer , Hesse Christian , Pelz Oliver
Published: December 2019

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of δ
13
C values of palmitic acid (C
16:0
) with the δ
13
C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C
16:0
with
13
C when growing on glycerol. With the exception of
Burkholderia gladioli
, all microorganism showed depletion of
13
C in C
16:0
while incorporating the carbons of glucose, and most of them were enriched with
13
C from mannose, with the exception of
Pseudomonas fluorescens
and the Zygomycotina. Usually, the glycolipid fractions are depleted in
13
C compared to the phospholipid fractions. The δ
13
C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C
14:0
) was depleted of
13
C compared to palmitic acid (C
16:0
) while octadecanoic acid (C
18:0
) was enriched. These results are important for the calibration of a new method in which δ
13
C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.
Partial Characterization of theStreptomyces lividans xlnBPromoter and Its Use for Expression of a Thermostable Xylanase fromThermotoga maritima
Authors: Chen Chih-Cheng , Westpheling Janet
Published: December 2019

Xylanase activity assays were used to screen aStreptomyces coelicolorgenomic library inEscherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) ofS. lividans(GenBank accession no.M64552) was identified. The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of theS. coelicolor xlnBgene and thexylEreporter gene. Transcription from thexlnBpromoter was found to be induced by xylan and repressed by glucose. A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays. Analysis of deletions of the promoter identified a region required for glucose repression. By using the transcriptional and protein localization signals of theStreptomyces xlnBgene, an in-frame translational fusion between the end of thexlnBsignal sequence and the ATG of theThermotoga maritima xynAgene was constructed. ThexynAgene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp. ThexlnB-xynAgene fusion was expressed inStreptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells.
Detection of Infectious Enteroviruses, Enterovirus Genomes, Somatic Coliphages, and
Bacteroides fragilis
Phages in Treated Wastewater
Authors: Gantzer C. , Maul A. , Audic J. M. , Schwartzbrod L.
Published: December 2019

In this study, three types of treated wastewater were tested for infectious enteroviruses, the enterovirus genome, somatic coliphages, and
Bacteroides fragilis
phages. The aim of this work was to determine whether the presence of the two types of bacteriophages or of the enterovirus genome was a good indicator of infectious enterovirus contamination. The enterovirus genome was detected by reverse transcription-polymerase chain reaction. Infectious enteroviruses were quantified by cell culturing (BGM cells), and the bacteriophages were quantified by plaque formation on the host bacterium (
Escherichia coli
or
B. fragilis
) in agar medium. Forty-eight samples of treated wastewater were analyzed. Sixteen samples had been subjected to a secondary treatment for 8 to 12 h (A), 16 had been subjected to a secondary treatment for 30 h (B1), and 16 had been subjected to both secondary and tertiary treatments (B2). The mean concentrations of somatic coliphages were 4.9 × 10
4
PFU · liter
−1
for treatment line A, 9.8 × 10
3
PFU · liter
−1
for B1, and 1.4 × 10
3
PFU · liter
−1
for B2, with all the samples testing positive (100%). The mean concentrations of
B. fragilis
phages were 1.7 × 10
3
PFU · liter
−1
for A (100% positive samples), 17 to 24 PFU · liter
−1
for B1 (44% positive samples), and 0.8 to 13 PFU · liter
−1
for B2 (6% positive samples). The mean concentrations of infectious enteroviruses were 4 most probable number of cytopathogenic units (MPNCU) · liter
−1
for A (31% positive samples) and <1 MPNCU · liter
−1
for B1 and B2 (0% positive samples). The percentages of samples testing positive for the enterovirus genome were 100% for A, 56% for B1, and 19% for B2. The percentages of samples testing positive for the enterovirus genome were significantly higher than those for infectious enteroviruses. This finding may have been due to the presence of noninfectious enteroviruses or to the presence of infectious enteroviruses that do not multiply in BGM cell cultures. However, under our experimental conditions, nondetection of the genome implies the absence of infectious viruses. There was a significant correlation between the concentration of somatic coliphages or
B. fragilis
phages and the presence of infectious enteroviruses or the presence of the enterovirus genome. However, the somatic coliphage concentration did not lead to fluctuations in the infectious enterovirus concentration, whereas the
B. fragilis
phage concentration did.
Identification and Quantification of Toxic Chemicals by Use of
Escherichia coli
Carrying
lux
Genes Fused to Stress Promoters
Authors: Ben-Israel Oren , Ben-Israel Haggit , Ulitzur Shimon
Published: December 2019

The
luxCDABE
bioluminescence genes of the
Vibrio fischeri lux
system have been used as a reporter system for different stress and regulatory promoters of
Escherichia coli
. Selected
E. coli
strains carrying
lux
genes fused to different promoters were exposed to various toxic chemicals, and the recorded luminescence was used for the characterization of the biologic signature of each compound. Analysis of these data with the aid of a proper algorithm allowed quantitative and qualitative assessment of toxic chemicals. Of the 25 tested chemicals, 23 were identified by this novel strategy in a 3-h procedure. This system can also be adapted for the identification of simple mixtures of toxic agents when the biologic signatures of the individual compounds are known. This biologic recognition strategy also provides a tool for evaluating the degree of similarity between the modes of action of different toxic agents.
Cyt1Aa Protein of
Bacillus thuringiensis
Is Toxic to the Cottonwood Leaf Beetle,
Chrysomela scripta
, and Suppresses High Levels of Resistance to Cry3Aa
Authors: Federici Brian A. , Bauer Leah S.
Published: December 2019

The insecticidal activity of
Bacillus thuringiensis
is due primarily to Cry and Cyt proteins. Cry proteins are typically toxic to lepidopterous, coleopterous, or dipterous insects, whereas the known toxicity of Cyt proteins is limited to dipterans. We report here that a Cyt protein, Cyt1Aa, is also highly toxic to the cottonwood leaf beetle,
Chrysomela scripta
, with a median lethal concentration of 2.5 ng/mm
2
of leaf surface for second-instar larvae. Additionally, we show that Cyt1Aa suppresses resistance to Cry3Aa greater than 5,000-fold in
C. scripta
, a level only partially overcome by Cry1Ba due to cross-resistance. Studies of the histopathology of
C. scripta
larvae treated with Cyt1Aa revealed disruption and sloughing of midgut epithelial cells, indicating that its mechanism of action against
C. scripta
is similar to that observed in mosquito and blackfly larvae. These novel properties suggest that Cyt proteins may have an even broader spectrum of activity against insects and, owing to their different mechanism of action in comparison to Cry proteins, might be useful in managing resistance to Cry3 and possibly other Cry toxins used in microbial insecticides and transgenic plants.
Implications of rRNA Operon Copy Number and Ribosome Content in the Marine Oligotrophic Ultramicrobacterium
Sphingomonas
sp. Strain RB2256
Authors: Fegatella Fitri , Lim Julianne , Kjelleberg Staffan , Cavicchioli Ricardo
Published: December 2019

Sphingomonas
sp. strain RB2256 is a representative of the dominant class of ultramicrobacteria that are present in marine oligotrophic waters. In this study we examined the rRNA copy number and ribosome content of RB2256 to identify factors that may be associated with the relatively low rate of growth exhibited by the organism. It was found that RB2256 contains a single copy of the rRNA operon, in contrast to
Vibrio
spp., which contain more than eight copies. The maximum number of ribosomes per cell was observed during mid-log phase; however, this maximum content was low compared to those of faster-growing, heterotrophic bacteria (approximately 8% of the maximum ribosome content of
Escherichia coli
with a growth rate of 1.5 h
−1
). The low number of ribosomes per cell appears to correlate with the low rate of growth (0.16 to 0.18 h
−1
) and the presence of a single copy of the rRNA operon. However, on the basis of cell volume, RB2256 appears to have a higher concentration of ribosomes than
E. coli
(approximately double that of
E. coli
with a growth rate of 1.5 h
−1
). Ribosome numbers reached maximum levels during mid-log-phase growth but decreased rapidly to 10% of maximum during late log phase through 7 days of starvation. The cells in late log phase and at the onset of starvation displayed an immediate response to a sudden addition of excess glucose (3 mM). This result demonstrates that a ribosome content 10% of maximum is sufficient to allow cells to immediately respond to nutrient upshift and achieve maximum rates of growth. These data indicate that the bulk of the ribosome pool is not required for protein synthesis and that ribosomes are not the limiting factor contributing to a low rate of growth. Our findings show that the regulation of ribosome content, the number of ribosomes per cell, and growth rate responses in RB2256 are fundamentally different from those characteristics in fast-growing heterotrophs like
E. coli
and that they may be characteristics typical of oligotrophic ultramicrobacteria.
Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of
Cryptosporidium parvum
Oocysts
Authors: Brush Charles F. , Walter Michael F. , Anguish Lynne J. , Ghiorse William C.
Published: December 2019

Surface properties of
Cryptosporidium parvum
oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m
2
V
−1
s
−1
over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (ς = 0.36) than that of oocysts stored in DI without antibiotics (ς = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m
2
V
−1
s
−1
at pH 2.4 to −3.2 × 10
−8
m
2
V
−1
s
−1
at pH 10 (ς = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter
−1
. Two-week-old oocysts exhibited strong adhesion (∼85%) at ionic strengths of 0 to 10 mmol liter
−1
and moderate adhesion (∼20%) at ionic strengths of 20 to 95 mmol liter
−1
. Two-month-old oocysts exhibited high adhesion (∼60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified
C. parvum
oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.
Sedimentation of Free and Attached
Cryptosporidium
Oocysts and
Giardia
Cysts in Water
Authors: Medema G. J. , Schets F. M. , Teunis P. F. M. , Havelaar A. H.
Published: December 2019

Experimental analysis of the sedimentation velocity of
Cryptosporidium parvum
oocysts and
Giardia lamblia
cysts was compared with mathematical description of their sedimentation velocities by using measurements of (oo)cyst size and density and the density and viscosity of the sedimentation medium to determine if the sedimentation kinetics of freely suspended oocysts of
C. parvum
and cysts of
G. lamblia
can be described by Stokes’ law. The theoretically calculated sedimentation kinetics showed a good agreement with the experimentally observed kinetics. Both showed a decline in sedimentation velocity over time, caused primarily by variation in (oo)cyst density. The initial apparent sedimentation velocities in Hanks balanced salt solution at 23°C was 0.35 μm · s
−1
for oocysts and 1.4 μm · s
−1
for cysts. (Oo)cysts that enter the surface water environment by discharges of biologically treated sewage may be attached to sewage particles, and this will affect their sedimentation kinetics. Therefore, (oo)cysts were mixed with settled secondary effluent. (Oo)cysts readily attached to the (biological) particles in effluent; 30% of both cysts and oocysts attached during the first minutes of mixing, and this fraction increased to approximately 75% after 24 h. The sedimentation velocity of (oo)cysts attached to secondary effluent particles increased with particle size and was (already in the smallest size fraction [1 to 40 μm]) determined by the sedimentation kinetics of the effluent particles. The observed sedimentation velocities of freely suspended (oo)cysts are probably too low to cause significant sedimentation in surface water or reservoirs. However, since a significant proportion of both cysts and oocysts attached readily to organic biological particles in secondary effluent, sedimentation of attached (oo)cysts after discharge into surface water will probably be a significant factor in the environmental ecology of
C. parvum
and
G. lamblia
. Attachment to particles influences not only sedimentation of (oo)cysts in surface water but also their behavior in drinking water treatment processes.
Antifungal Metabolites (Monorden, Monocillin IV, and Cerebrosides) from
Humicola fuscoatra
Traaen NRRL 22980, a Mycoparasite of
Aspergillus flavus
Sclerotia
Authors: Wicklow Donald T. , Joshi Biren K. , Gamble William R. , Gloer James B. , Dowd Patrick F.
Published: December 2019

The mycoparasite
Humicola fuscoatra
NRRL 22980 was isolated from a sclerotium of
Aspergillus flavus
that had been buried in a cornfield near Tifton, Ga. When grown on autoclaved rice, this fungus produced the antifungal metabolites monorden, monocillin IV, and a new monorden analog. Each metabolite produced a clear zone of inhibition surrounding paper assay disks on agar plates seeded with conidia of
A. flavus
. Monorden was twice as inhibitory to
A. flavus
mycelium extension (MIC > 28 μg/ml) as monocillin IV (MIC > 56 μg/ml). Cerebrosides C and D, metabolites known to potentiate the activity of cell wall-active antibiotics, were separated from the ethyl acetate extract but were not inhibitory to
A. flavus
when tested as pure compounds. This is the first report of natural products from
H. fuscoatra
.
Quantification of 16S rRNAs in Complex Bacterial Communities by Multiple Competitive Reverse Transcription-PCR in Temperature Gradient Gel Electrophoresis Fingerprints
Authors: Felske Andreas , Akkermans Antoon D. L. , De Vos Willem M.
Published: December 2019

A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.
Genetic Characterization of
pepP
, Which Encodes an Aminopeptidase P Whose Deficiency Does Not Affect
Lactococcus lactis
Growth in Milk, Unlike Deficiency of the X-Prolyl Dipeptidyl Aminopeptidase
Authors: Matos J. , Nardi M. , Kumura H. , Monnet V.
Published: December 2019

We sequenced the
pepP
gene of
Lactococcus lactis
, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition. PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.
Metal Toxicity Reduction in Naphthalene Biodegradation by Use of Metal-Chelating Adsorbents
Authors: Malakul Pomthong , Srinivasan Keeran R. , Wang Henry Y.
Published: December 2019

A model system comprising microbial degradation of naphthalene in the presence of cadmium has been developed to evaluate metal toxicity associated with polyaromatic hydrocarbon biodegradation and its reduction by the use of unmodified and surfactant-modified clays in comparison with a commercially available chelating resin (Chelex 100; Bio-Rad). The toxicity of cadmium associated with naphthalene biodegradation was shown to be reduced significantly by using the modified-clay complex and Chelex resin, while unmodified clay has no significant impact on this reduction. The degree of metal toxicity reduction can be quantitatively related to the metal adsorption characteristics of these adsorbents, such as adsorption capacity and selectivity.
Response of
Salmonella choleraesuis
LT2 Spheroplasts and Permeabilized Cells to the Bacteriocin AS-48
Authors: Abriouel H. , Valdivia E. , Gálvez A. , Maqueda M.
Published: December 2019

The bacteriocin AS-48 was not active against intact cells of
Salmonella choleraesuis
LT2 at neutral pH, but it was very effective on spheroplasts, suggesting that the outer membrane (OM) acts as a protective barrier. Cells sublethally injured by heat or treated with OM-permeabilizing agents (i.e., EDTA and Tris) became sensitive to AS-48. The combination of two or more treatments decreased the amount of AS-48 required for cell killing. The activity of AS-48 against heat-injured cells did not change significantly in the pH range of 4.0 to 8.0. AS-48 showed bactericidal activity against intact cells of
Salmonella
at pH 4.0. The potency of AS-48 increased greatly when the bacteriocin was dissolved at pH 9.0.
Effect of Low-Osmolality Nutrient Media on Growth and Culturability of
Campylobacter
Species
Authors: Reezal Abdul , McNeil Brian , Anderson John G.
Published: December 2019

The growth and culturability of
Campylobacter jejuni
NCTC 11351 and other campylobacters were examined in media having different osmolalities at a range of temperatures (4, 25, and 42°C). The medium osmolalities used ranged from the osmolality of full-strength nutrient medium (modified campylobacter broth having an osmolality of around 254 mosmol) down to 96 mosmol. The following two methods were used to produce media having different osmolalities: dilution of the nutrient medium with distilled water and reformulation of the medium such that the concentrations of various osmolytes were altered while the nutrient content of the medium was unchanged. The results obtained with the two experimental methods were similar, indicating that there was an osmotic threshold effect, such that none of the campylobacters examined (
C. jejuni
NCTC 11351 and ATCC 33291,
Campylobacter lari
, and
Campylobacter coli
) grew in media having osmolalities around 130 mosmol and at temperatures below at 42°C. Conversely, growth occurred in media having osmolalities of around 175 mosmol and above. Osmolar concentrations can be expressed in terms of osmolarity or osmolality. Osmolality is easier to evaluate, is the more commonly used term, and was used in the current study. In nutrient media having low osmolalities (i.e., 130 mosmol and below), the number of CFUs per milliliter declined rapidly regardless of the temperature, and no cells were recovered after 24 h. However, at nongrowth temperatures (25 and 4°C) in higher-osmolality media (175 mosmol and above) a significant population was recovered throughout the experiment (up to 96 h). In low-osmolality nutrient media, the cellular morphology was principally coccoid, while in the early stages of growth in full-strength media the morphology was predominantly rodlike. We propose that the formation of coccoid cells in these experiments was the result of osmotic stress in low-osmolality media. This osmotic effect was apparent regardless of the osmolyte used to reformulate the medium (NaCl, KCl, Na
2
SO
4
, NH
4
Cl, and glucose were used).
Use of
13
C Nuclear Magnetic Resonance and Gas Chromatography To Examine Methionine Catabolism by Lactococci
Authors: Gao Song , Mooberry Ed S. , Steele James L.
Published: December 2019

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways,
13
C nuclear magnetic resonance (NMR) performed with uniformly enriched [
13
C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of
Lactococcus lactis
was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the
13
C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.
An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area
Authors: Corroler D. , Mangin I. , Desmasures N. , Gueguen M.
Published: December 2019

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a
Lactococcus lactis
subsp.
lactis
phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of
L. lactis
subsp.
lactis
or
L. lactis
subsp.
cremoris
. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of
L. lactis
was determined; 80% of the Bocage Falaisien strains were members of
L. lactis
subsp.
lactis
, and 30.5% of the Bessin strains were members of
L. lactis
subsp.
lactis
. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of
L. lactis
subsp.
cremoris
was as high as 66%. The
L. lactis
subsp.
lactis
strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of
L. lactis
subsp.
lactis
fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some
L. lactis
subsp.
lactis
strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of
L. lactis
subsp.
lactis
strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.
Cloning, Expression, and Chromosomal Stabilization of thePropionibacterium shermaniiProline Iminopeptidase Gene (pip) for Food-Grade Application inLactococcus lactis
Authors: Leenhouts Kees , Bolhuis Albert , Boot Johan , Deutz Inge , Toonen Marjolein , Venema Gerard , Kok Jan , Ledeboer Aat
Published: December 2019

Proline iminopeptidase produced byPropionibacterium shermaniiplays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed inEscherichia coliandLactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements thanP. shermanii. The levels of expression of thepipgene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter inL. lactisor the inducibletacpromoter inE. coli. Stable replication of the rolling-circle replicating (rcr) plasmid, used to expresspipinL. lactis, could only be obtained by providing therepAgene intrans. Upon the integration ofpip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose. The multicopy integrants demonstrated a high degree of stability in the presence of glucose. This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes inL. lactis.
Strain-Specific Identification of Probiotic
Lactobacillus rhamnosus
with Randomly Amplified Polymorphic DNA-Derived PCR Primers
Authors: Tilsala-Timisjärvi Anu , Alatossava Tapani
Published: December 2019

In the present work, strain-specific PCR primers for
Lactobacillus rhamnosus
Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11
L. rhamnosus
strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to
L. rhamnosus
Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11
Lactobacillus
species and 11
L. rhamnosus
strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.
Metabolism of Benzene, Toluene, and Xylene Hydrocarbons in Soil
Authors: Tsao C.-W. , Song H.-G. , Bartha R.
Published: December 2019

Enrichment cultures obtained from soil exposed to benzene, toluene, and xylene (BTX) mineralized benzene and toluene but cometabolized only xylene isomers, forming polymeric residues. This observation prompted us to investigate the metabolism of14C-labeled BTX hydrocarbons in soil, either individually or as mixtures. BTX-supplemented soil was incubated aerobically for up to 4 weeks in a sealed system that automatically replenished any O2consumed. The decrease in solvent vapors and the production of14CO2were monitored. At the conclusion of each experiment,14C distribution in solvent-extractable polymers, biomass, and humic material was determined, obtaining14C mass balances of 85 to 98%. BTX compounds were extensively mineralized in soil, regardless of whether they were presented singly or in combinations. No evidence was obtained for the formation of solvent-extractable polymers from xylenes in soil, but14C distribution in biomass (5 to 10%) and humus (12 to 32%) was unusual for all BTX compounds and especially for toluene and the xylenes. The results suggest that catechol intermediates of BTX degradation are preferentially polymerized into the soil humus and that the methyl substituents of the catechols derived from toluene and especially from xylenes enhance this incorporation. In contrast to inhibitory residues formed from xylene cometabolism in culture, the humus-incorporated xylene residues showed no significant toxicity in the Microtox assay.
Carbon Monoxide Oxidation by Bacteria Associated with the Roots of Freshwater Macrophytes
Authors: Rich Jeremy J. , King G. M.
Published: December 2019

The potential rates and control of aerobic root-associated carbon monoxide (CO) consumption were assessed by using excised plant roots from five common freshwater macrophytes. Kinetic analyses indicated that the maximum potential uptake velocities for CO consumption ranged from 0.4 to 2.7 μmol of CO g (dry weight)
−1
h
−1
for the five species. The observed rates were comparable to previously reported rates of root-associated methane uptake. The apparent half-saturation constants for CO consumption ranged from 50 to 370 nM CO; these values are considerably lower than the values obtained for methane uptake. The CO consumption rates reached maximum values at temperatures between 27 and 32°C, and there was a transition to CO production at ≥44°C, most likely as a result of thermochemical organic matter decomposition. Incubation of roots with organic substrates (e.g., 5 mM syringic acid, glucose, alanine, and acetate) dramatically reduced the rate of CO consumption, perhaps reflecting a shift in metabolism by facultative CO oxidizers. Based on responses to a suite of antibiotics, most of the CO consumption (about 90%) was due to eubacteria rather than fungi or other eucaryotes. Based on the results of acetylene inhibition experiments, methanotrophs and ammonia oxidizers were not active CO consumers.
Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi
Authors: Vierheilig Horst , Coughlan Andrew P. , Wyss Urs , Piché Yves
Published: December 2019

We developed a reliable, inexpensive, and simple method for staining arbuscular-mycorrhizal fungal colonizations in root tissues. Apart from applications in research, this nontoxic, high-quality staining method also could be of great utility in teaching exercises. After adequate clearing with KOH, an ink-vinegar solution successfully stained all fungal structures, rendering them clearly visible.
Construction of a Bioluminescent Reporter Strain To Detect Polychlorinated Biphenyls
Authors: Layton A. C. , Muccini M. , Ghosh M. M. , Sayler G. S.
Published: December 2019

A bioluminescent reporter strain,
Ralstonia eutropha
ENV307(pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.
Production of Monoclonal Antibodies Specific for the i and
1,2
Flagellar Antigens of
Salmonella typhimurium
and Characterization of Their Respective Epitopes
Authors: de Vries N. , Zwaagstra K. A. , Huis in’t Veld J. H. J. , van Knapen F. , van Zijderveld F. G. , Kusters J. G.
Published: December 2019

Salmonella typhimurium
expresses two antigenically distinct flagellins, each containing a different H antigen (
i
and
1,2
), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the
fliC
(H:
i
) and
fljB
(H:
1,2
) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:
i
and 102 amino acids for H:
1,2
, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting
S. typhimurium
-specific antibody.
A Simple and Reliable Method for Hybridization of Homothallic Wine Strains ofSaccharomyces cerevisiae
Authors: Ramírez Manuel , Peréz Francisco , Regodón José A.
Published: December 2019

A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains.
The Bactericidal Activity of Pediocin PA-1 Is Specifically Inhibited by a 15-mer Fragment That Spans the Bacteriocin from the Center toward the C Terminus
Authors: Fimland Gunnar , Jack Ralph , Jung Günther , Nes Ingolf F. , Nissen-Meyer Jon
Published: December 2019

A 15-mer peptide fragment derived from pediocin PA-1 (from residue 20 to residue 34) specifically inhibited the bactericidal activity of pediocin PA-1. The fragment did not inhibit the pediocin-like bacteriocins sakacin P, leucocin A, and curvacin A to nearly the same extent as it inhibited pediocin PA-1. Enterocin A, however, was also significantly inhibited by this fragment, although not as greatly as pediocin PA-1. This is consistent with the fact that enterocin A contains the longest continuous sequence identical to that of pediocin PA-1 in the region spanned by the fragment. The fragment inhibited pediocin PA-1 to a much greater extent than did the other 29 possible 15-mer fragments that span pediocin PA-1. The results suggest that the fragment—by interacting with the target cells and/or pediocin PA-1—interferes specifically with pediocin-target cell interaction.
Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom
Korarchaeota
Authors: Brunk Clifford F. , Eis Nicole
Published: December 2019

Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.
A New 4-Nitrotoluene Degradation Pathway in a
Mycobacterium
Strain
Authors: Spiess Tilmann , Desiere Frank , Fischer Peter , Spain Jim C. , Knackmuss Hans-Joachim , Lenke Hiltrud
Published: December 2019

Mycobacterium
sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the
Mycobacterium
strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-
m
-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-
m
-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-
m
-cresol, observed during growth of the
Mycobacterium
strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (λ
max
, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-
m
-cresol by
meta
ring cleavage.
High-Level Production of Recombinant Human Parathyroid Hormone 1-34
Authors: Suzuki Yuji , Yabuta Masayuki , Ohsuye Kazuhiro
Published: December 2019

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified
Escherichia coli
β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the
E. coli
total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.
Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from
Trichoderma reesei
QM9414
Authors: Okada Hirofumi , Tada Kohji , Sekiya Tadashi , Yokoyama Kengo , Takahashi Akinori , Tohda Hideki , Kumagai Hiromichi , Morikawa Yasushi
Published: December 2019

We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-β-1,4-glucanase) gene from
Trichoderma reesei
QM9414. The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide. The deduced protein sequence has significant homologies with family H endo-β-1,4-glucanases. The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion. Northern blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by glucose. The protein has been expressed as a mature form in
Escherichia coli
and as secreted forms in
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
under the control of
tac
, alcohol dehydrogenase (
ADH1
), and human cytomegalovirus promoters, respectively. The
S. cerevisiae
and
Schizosaccharomyces pombe
recombinant strains showed strong cellulolytic activities on agar plates containing carboxymethyl cellulose. The
E. coli
strain expressed small amounts of EG III in an active form and large amounts of EG III in an inactive form. The molecular masses of the recombinant EG IIIs were estimated to be 25, 28, and 29 kDa for
E. coli
,
S. cerevisiae
, and
Schizosaccharomyces pombe
, respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Parts of the yeast recombinant EG IIIs decreased their molecular masses to 25 kDa after treatment with endoglycosidase H and α-mannosidase, suggesting that they are N glycosylated at least partly.
Enterococcus faecalis
Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants
Authors: Marcinek Herbert , Wirth Reinhard , Muscholl-Silberhorn Albrecht , Gauer Matthias
Published: December 2019

The ability of
Enterococcus faecalis
to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn
916
(a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of
E. faecalis
under natural conditions was, compared to that under laboratory conditions, at least 10
5
-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn
916
. In no case was transfer from
E. faecalis
to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of
E. faecalis
in the municipal sewage water treatment plant of the city of Regensburg ranged from 10
5
to 10
8
events per 4 h, indicating that gene transfer should take place under natural conditions.
A Rapid, Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water
Authors: Messer James W. , Dufour Alfred P.
Published: December 2019

A two-step membrane filter (MF) method with mE medium, upon which the membrane must be incubated for 48 h and then transferred to a substrate medium to differentiate enterococci, is recommended by the U.S. Environmental Protection Agency to measure enterococci in fresh and marine recreational waters. The original mE medium was modified by reducing the triphenyltetrazolium chloride from 0.15 to 0.02 g/liter and adding 0.75 g of indoxyl β-
d
-glucoside per liter. The new MF medium, mEI medium, detected levels of enterococci in 24 h comparable to those detected by the original mE medium in 48 h, with the same level of statistical confidence. In addition, the use of mEI medium eliminated the need to transfer the membrane to a substrate medium to differentiate enterococci from other genera of the fecal streptococcal group. Colonies from mEI medium were examined to determine the rates of false-positive and false-negative occurrences. mEI medium had a false-positive rate of 6.0% and a false-negative rate of 6.5%. Interlaboratory testing of the MF method with mEI medium demonstrated that the relative reproducibility standard deviations among laboratories ranged from 2.2% for marine water to 18.9% for freshwater. The comparative recovery studies, specificity determinations, and multilaboratory evaluation indicated that mEI medium has analytical performance characteristics equivalent to those of mE medium. The simplicity of use and decreased incubation time with mEI medium will facilitate the detection and quantification of enterococci in fresh and marine recreational waters.
Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments
Authors: Ogunseitan O. A.
Published: December 2019

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with
Pseudomonas aeruginosa
PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (
r
2
= 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10
2
CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (10
2
to 10
8
CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.
Microbial Conversion Products of Leptomycin B
Authors: Kuhnt Michaela , Bitsch Francis , Ponelle Monique , Sanglier Jean-Jacques , Wang Ying , Wolff Barbara
Published: December 2019

Leptomycin B (LMB), a secondary metabolite produced by
Streptomyces
sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of >5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with
Aspergillus flavus
ATCC 9170 and
Emericella unguis
ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively.
Streptomyces rimosus
ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.