bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of Chemotaxis to Naphthalene by
Pseudomonas putida
G7
Authors: Marx Randall B. , Aitken Michael D.
Published: December 2019

The capillary assay was used to quantify the chemotactic response of
Pseudomonas putida
G7 to naphthalene. Experiments were conducted in which the cell concentration in the assay chamber, the naphthalene concentration in the capillary, or the incubation time was varied. Data from these experiments were evaluated with a model that accounted for the effect of diffusion on the distribution of substrate and the transport of cells from the chamber through the capillary orifice. By fitting a numerical solution of this model to the data, it was possible to determine the chemotactic sensitivity coefficient, χ
0
. The mean of the best-fit values for χ
0
from the three types of experiments was 7.2 × 10
−5
cm
2
/s. A less computationally intensive model based on earlier approaches that ignore cell transport in the chamber resulted in χ
0
values that were approximately three times higher. The models evaluated in the present study could simulate the results of capillary assays only at low chamber cell concentrations, for which the effect of consumption on the distribution of substrate was negligible. Results from this work suggest that it is possible to use the capillary assay to quantify taxis towards environmentally relevant chemoeffectors that have low aqueous solubility.
Changes in Membrane Fatty Acid Composition of
Pediococcus
sp. Strain NRRL B-2354 in Response to Growth Conditions and Its Effect on Thermal Resistance
Authors: Annous Bassam A. , Kozempel Michael F. , Kurantz Michael J.
Published: December 2019

Membrane fatty acid composition and thermal resistance (
D
value) of
Pediococcus
sp. were determined for mid-exponential-phase (ME) and stationary-phase (ST) cells grown in tryptic soy broth (TSB) and tryptone-glucose-yeast extract (TGY) at 28 and 37°C. As the cells entered the stationary phase of growth, the unsaturated fatty acid, C

18:1 
n
11c

, produced during the exponential phase of growth was converted to its cyclic form, C
19:0 Δ9c
. This shift in membrane fatty acid composition was accompanied by an increase in the
D
values of this bacterium. Data from this study suggest that the membrane fatty acid composition of
Pediococcus
sp. is dependent on the growth conditions and that membrane fatty acid composition plays a critical role in thermal resistance. Thermal inactivation curves of
Pediococcus
sp. cells grown in TGY at 28°C indicated the presence of a cell population that is heterogeneous in thermal resistance. The growth of this bacterium in TGY at 37°C and in TSB at 28 and 37°C resulted in cell populations that were uniform in thermal resistance with a lag time for thermal inactivation. Thermal inactivation curves of ME and ST cultures were similar. The data presented here suggest that the cell population’s uniformity of thermal inactivation is independent of the growth phase of the culture.
Identification and Sequencing of β-Myrcene Catabolism Genes from
Pseudomonas
sp. Strain M1
Authors: Iurescia Sandra , Marconi Andrea M. , Tofani Daniela , Gambacorta Augusto , Paternò Annalisa , Devirgiliis Chiara , van der Werf Mariët J. , Zennaro Elisabetta
Published: December 2019

The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a
Pseudomonas
sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (
E
)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named
myrA
,
myrB
,
myrC
, and
myrD
, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively.
myrA
,
myrB
, and
myrC
are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The
myrD
gene starts 224 nt upstream of
myrA
and is divergently transcribed. The
myrB
sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the
myrB
gene, in complete agreement with the accumulation of (
E
)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in
Pseudomonas
sp. strain M1.
Characterization and Identification of
Pediococcus
Species Isolated from Forage Crops and Their Application for Silage Preparation
Authors: Cai Yimin , Kumai Sumio , Ogawa Masuhiro , Benno Yoshimi , Nakase Takashi
Published: December 2019

Pediococcus
species isolated from forage crops were characterized, and their application to silage preparation was studied. Most isolates were distributed on forage crops at low frequency. These isolates could be divided into three (A, B, and C) groups by their sugar fermentation patterns. Strains LA 3, LA 35, and LS 5 are representative isolates from groups A, B, and C, respectively. Strains LA 3 and LA 35 had intragroup DNA homology values above 93.6%, showing that they belong to the species
Pediococcus acidilactici
. Strain LS 5 belonged to
Pediococcus pentosaceus
on the basis of DNA-DNA relatedness. All three of these strains and strain SL 1 (
Lactobacillus casei
, isolated from a commercial inoculant) were used as additives to alfalfa and Italian ryegrass silage preparation at two temperatures (25 and 48°C). When stored at 25°C, all of the inoculated silages were well preserved and exhibited significantly (
P
< 0.05) reduced fermentation losses compared to that of their control in alfalfa and Italian ryegrass silages. When stored at 48°C, silages inoculated with strains LA 3 and LA 35 were also well preserved, with a significantly (
P
< 0.05) lower pH, butyric acid and ammonia-nitrogen content, gas production, and dry matter loss and significantly (
P
< 0.05) higher lactate content than the control, but silages inoculated with LS 5 and SL 1 were of poor quality.
P. acidilactici
LA 3 and LA 35 are considered suitable as potential silage inoculants.
Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and Metabolite Production
Authors: Park D. H. , Laivenieks M. , Guettler M. V. , Jain M. K. , Zeikus J. G.
Published: December 2019

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H
2
-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (−1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by
Actinobacillus succinogenes
or by mixed methanogenic cultures. The
A. succinogenes
mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO
2
. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.
16S rRNA Gene Sequence Analysis of
Photobacterium damselae
and Nested PCR Method for Rapid Detection of the Causative Agent of Fish Pasteurellosis
Authors: Osorio Carlos R. , Collins Matthew D. , Toranzo Alicia E. , Barja Juan L. , Romalde Jesús L.
Published: December 2019

The causative agent of fish pasteurellosis, the organism formerly known as
Pasteurella piscicida
, has been reclassified as
Photobacterium damselae
subsp.
piscicida
on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as
Photobacterium damselae
subsp.
damselae
(formerly
Vibrio damselae
). Since reassignment of
P. damselae
subsp.
piscicida
was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of
P. damselae
based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of
P. damselae
subsp.
piscicida
, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.
Physiological Adaptations Involved in Alkane Assimilation at a Low Temperature by
Rhodococcus
sp. Strain Q15
Authors: Whyte L. G. , Slagman S. J. , Pietrantonio F. , Bourbonnière L. , Koval S. F. , Lawrence J. R. , Inniss W. E. , Greer C. W.
Published: December 2019

We examined physiological adaptations which allow the psychrotroph
Rhodococcus
sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5°C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5°C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [β-
d
-Gal-(1-3)-
d
-GlcNAc and α-
l
-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5°C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5°C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.
Mechanism of Pyrite Dissolution in the Presence of
Thiobacillus ferrooxidans
Authors: Fowler T. A. , Holmes P. R. , Crundwell F. K.
Published: December 2019

In spite of the environmental and commercial interests in the bacterial leaching of pyrite, two central questions have not been answered after more than 35 years of research: does
Thiobacillus ferrooxidans
enhance the rate of leaching above that achieved by ferric sulfate solutions under the same conditions, and if so, how do the bacteria affect such an enhancement? Experimental conditions of previous studies were such that the concentrations of ferric and ferrous ions changed substantially throughout the course of the experiments. This has made it difficult to interpret the data obtained from these previous works. The aim of this work was to answer these two questions by employing an experimental apparatus designed to maintain the concentrations in solution at a constant value. This was achieved by using the constant redox potential apparatus described previously (P. I. Harvey, and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586–2592, 1997; T. A. Fowler, and F. K. Crundwell, Appl. Environ. Microbiol. 64:3570–3575, 1998). Experiments were conducted in both the presence and absence of
T. ferrooxidans
, maintaining the same conditions in solution. The rate of dissolution of pyrite with bacteria was higher than that without bacteria at the same concentrations of ferrous and ferric ions in solution. Analysis of the dependence of the rate of leaching on the concentration of ferric ions and on the pH, together with results obtained from electrochemical measurements, provided clear evidence that the higher rate of leaching with bacteria is due to the bacteria increasing the pH at the surface of the pyrite.
Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils
Authors: Bruns Mary Ann , Stephen John R. , Kowalchuk George A. , Prosser James I. , Paul Eldor A.
Published: December 2019

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within
Nitrosospira
cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for
Nitrosospira
cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the
Nitrosospira
cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.
Isolation and Characterization of Proteolytic Ruminal Bacteria from Sheep and Goats Fed the Tannin-Containing Shrub Legume
Calliandra calothyrsus
Authors: McSweeney Christopher S. , Palmer Brian , Bunch Rowan , Krause Denis O.
Published: December 2019

Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed
Calliandra calothyrsus
(3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97.6%) to
Clostridium botulinum
NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin.
Hemolysis, Toxicity, and Randomly Amplified Polymorphic DNA Analysis of
Stachybotrys chartarum
Strains
Authors: Vesper Stephen J. , Dearborn Dorr G. , Yike Iwona , Sorenson W. G. , Haugland Richard A.
Published: December 2019

Stachybotrys chartarum
is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of
S. chartarum
isolated from case (
n
= 8) or control (
n
= 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23°C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep’s blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23°C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37°C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 μg of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.
Comparison of Sensitivity of Immunofluorescent Microscopy to That of a Combination of Immunofluorescent Microscopy and Immunomagnetic Separation for Detection ofCryptosporidium parvumOocysts in Adult Bovine Feces
Authors: Pereira Maria das Graças C. , Atwill Edward R. , Jones Ted
Published: December 2019

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations ofCryptosporidium parvumoocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.
In Situ Detection of the
Clostridium botulinum
Type C
1
Toxin Gene in Wetland Sediments with a Nested PCR Assay
Authors: Williamson Judy L. , Rocke Tonie E. , Aiken Judd M.
Published: December 2019

A nested PCR was developed for detection of the
Clostridium botulinum
type C
1
toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C
1
toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of
C. botulinum
type C in wetland sediments and the sensitivity of the detection assay.
Characterization of Methylglyoxal Synthase from
Clostridium acetobutylicum
ATCC 824 and Its Use in the Formation of 1,2-Propanediol
Authors: Huang Ke-xue , Rudolph Frederick B. , Bennett George N.
Published: December 2019

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in
Clostridium acetobutylicum
ATCC 824. The enzyme was overexpressed in
Escherichia coli
and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The

K
m

and
V
max
values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min
−1
μg
−1
, respectively. When
E. coli
glycerol dehydrogenase was coexpressed with methylglyoxal synthase in
E. coli
BL21(DE3), 3.9 mM 1,2-propanediol was produced.
Determination of Total Protein Content of Bacterial Cells by SYPRO Staining and Flow Cytometry
Authors: Zubkov Mikhail V. , Fuchs Bernhard M. , Eilers Heike , Burkill Peter H. , Amann Rudolf
Published: December 2019

An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of
Bacteria
(an
Arcobacter
sp., a
Cytophaga
sp., an
Oceanospirillum
sp., a
Pseudoalteromonas
sp., and a
Vibrio
sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell
−1
(
r
2
= 0.93,
n
= 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell
−1
.
Characterization of Methanotrophic Bacterial Populations in Soils Showing Atmospheric Methane Uptake
Authors: Holmes Andrew J. , Roslev Peter , McDonald Ian R. , Iversen Niels , Henriksen Kaj , Murrell J. Colin
Published: December 2019

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the ∼1% · year
−1
increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with
14
CH
4
and analyzing a total soil monooxygenase gene library. Comparative analyses of [
14
C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the α subclass of the class
Proteobacteria
was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of “atmospheric methane oxidizers” should be greatly facilitated by use of biomarkers identified in this study.
Abr1, a Transposon-Like Element in the Genome of the Cultivated MushroomAgaricus bisporus(Lange) Imbach
Authors: Sonnenberg Anton S. M. , Baars Johan J. P. , Mikosch Thomas S. P. , Schaap Peter J. , Van Griensven Leo J. L. D.
Published: December 2019

A 300-bp repetitive element was found in the genome of the white button mushroom,Agaricus bisporus, and designatedAbr1. It is present in ∼15 copies per haploid genome in the commercial strain Horst U1. Analysis of seven copies showed 89 to 97% sequence identity. The repeat has features typical of class II transposons (i.e., terminal inverted repeats, subterminal repeats, and a target site duplication of 7 bp). The latter shows a consensus sequence. When used as probe on Southern blots,Abr1identifies relatively little variation within traditional and present-day commercial strains, indicating that most strains are identical or have a common origin. In contrast to these cultivars, high variation is found among field-collected strains. Furthermore, a remarkable difference in copy numbers ofAbr1was found betweenA. bisporusisolates with a secondarily homothallic life cycle and those with a heterothallic life cycle.Abr1is a type II transposon not previously reported in basidiomycetes and appears to be useful for the identification of strains within the speciesA. bisporus.
Production of Acylated Homoserine Lactones by Psychrotrophic Members of the
Enterobacteriaceae
Isolated from Foods
Authors: Gram Lone , Christensen Allan Beck , Ravn Lars , Molin Søren , Givskov Michael
Published: December 2019

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic
Enterobacteriaceae
strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that
N
-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10
6
CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of
Enterobacteriaceae
stored at 5°C under an N
2
atmosphere when mean cell densities increased to 10
6
CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous
Enterobacteriaceae
reached 10
6
CFU/g. This level of
Enterobacteriaceae
is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.
Activation Process of Dipteran-Specific Insecticidal Protein Produced by
Bacillus thuringiensis
subsp.
israelensis
Authors: Yamagiwa Masashi , Esaki Motoyuki , Otake Kanao , Inagaki Manabu , Komano Tohru , Amachi Teruo , Sakai Hiroshi
Published: December 2019

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in
Bacillus thuringiensis
subsp.
israelensis
. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione
S
-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of
Culex pipiens
, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of
C. pipiens
larvae had toxicity slightly lower than that of GST-60.
Genomic Characterization of Human and Environmental Polioviruses Isolated in Albania
Authors: Divizia Maurizio , Palombi Leonardo , Buonomo Ersilia , Donia Domenica , Ruscio Vito , Equestre Michele , Leno Luljeta , Panà Augusto , Degener Anna Marta
Published: December 2019

Between April and December 1996, a serious outbreak of poliomyelitis occurred in Albania; almost 140 subjects were involved, and the episode presented an unusually high mortality rate (12%). During the outbreak, water samples from the Lana River in Tirana, Albania, and stool samples from two cases of paralytic poliomyelitis were collected and analyzed for the presence of polioviruses. Six polioviruses were isolated from the environmental and human samples, according to standard methods. All the samples were characterized by partial genomic sequencing of 330 bases across the 5′ untranslated region (5′-UTR) (nucleotide positions 200 to 530) and of 300 bases across the VP1 region (nucleotide positions 2474 to 2774). Comparison of these sequences with those present in data banks permitted the identification of environmental isolates Lana A and Lana B as, respectively, a Sabin-like type 2 poliovirus and an intertypic recombinant poliovirus (Sabin-like type 2/wild type 1), both bearing a G instead of an A at nucleotide position 481. The two other environmental polioviruses were similar to the isolates from the paralytic cases. They were characterized by a peculiar 5′-UTR and by a VP1 region showing 98% homology with the Albanian epidemic type 1 isolates reported by other authors. This study confirms the environmental circulation in Albania of recombinant poliovirus strains, likely sustained by a massive vaccination effort and by the presence in the environment of a type 1 poliovirus, as isolated from the Lana River in Tirana about 2 months before the first case of symptomatic acute flaccid paralysis was reported in this town.
Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the
bph
-Encoded Catabolic Pathway of
Burkholderia
sp. Strain LB400
Authors: Seeger Michael , Zielinski Marco , Timmis Kenneth N. , Hofer Bernd
Published: December 2019

Burkholderia
sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its
bph
gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated
meta
or
para
carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the
ortho
position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of
ortho
-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the
bph
-encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.
Isolation and Characterization of
Methanomethylovorans hollandica
gen. nov., sp. nov., Isolated from Freshwater Sediment, a Methylotrophic Methanogen Able To Grow on Dimethyl Sulfide and Methanethiol
Authors: Lomans Bart P. , Maas Ronald , Luderer Rianne , Op den Camp Huub J. M. , Pol Arjan , van der Drift Chris , Vogels Godfried D.
Published: December 2019

A newly isolated methanogen, strain DMS1
T
, is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1
T
are methanol, methylamines, methanethiol (MT), and DMS, this organism is considered an obligately methylotrophic methanogen like most other DMS-degrading methanogens. Strain DMS1
T
differs from all other DMS-degrading methanogens, since it was isolated from a freshwater pond and requires NaCl concentrations (0 to 0.04 M) typical of the NaCl concentrations required by freshwater microorganisms for growth. DMS was degraded effectively only in a chemostat culture in the presence of low hydrogen sulfide and MT concentrations. Addition of MT or sulfide to the chemostat significantly decreased degradation of DMS. Transient accumulation of DMS in MT-amended cultures indicated that transfer of the first methyl group during DMS degradation is a reversible process. On the basis of its low level of homology with the most closely related methanogen,
Methanococcoides burtonii
(94.5%), its position on the phylogenetic tree, its morphology (which is different from that of members of the genera
Methanolobus
,
Methanococcoides
, and
Methanohalophilus
), and its salt tolerance and optimum (which are characteristic of freshwater bacteria), we propose that strain DMS1
T
is a representative of a novel genus. This isolate was named
Methanomethylovorans hollandica
. Analysis of DMS-amended sediment slurries with a fluorescence microscope revealed the presence of methanogens which were morphologically identical to
M. hollandica
, as described in this study. Considering its physiological properties,
M. hollandica
DMS1
T
is probably responsible for degradation of MT and DMS in freshwater sediments in situ. Due to the reversibility of the DMS conversion, methanogens like strain DMS1
T
can also be involved in the formation of DMS through methylation of MT. This phenomenon, which previously has been shown to occur in sediment slurries of freshwater origin, might affect the steady-state concentrations and, consequently, the total flux of DMS and MT in these systems.
Rapid Fluorescence Assessment of the Viability of Stressed
Lactococcus lactis
Authors: Bunthof Christine J. , van den Braak Sabina , Breeuwer Pieter , Rombouts Frank M. , Abee Tjakko
Published: December 2019

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using
Lactococcus lactis
subsp.
lactis
ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70°C very well. However, after treatment up to 60°C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50°C and was completely lost for those treated at 60°C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed
L. lactis
and has potential for physiological research and dairy applications related to lactic acid bacteria.
High Rates of Conjugation in Bacterial Biofilms as Determined by Quantitative In Situ Analysis
Authors: Hausner Martina , Wuertz Stefan
Published: December 2019

Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.
Development and Testing of a Microbiological Assay To Detect Residual Effects of Disinfectant on Hard Surfaces
Authors: Mariscal Alberto , Carnero-Varo Manuel , Gómez-Aracena Jorge , Fernández-Crehuet Joaquín
Published: December 2019

We describe a glucuronidase bioassay for detecting residual bactericidal activity from the use of disinfectants on hard surfaces; in this assay we used formaldehyde, ethanol, isopropanol, chlorine, and a commercial preparation containing 2-bromo-2-nitro-1,3-propanediol. Chlorine and the commercial preparation showed bactericidal activity (53.5% and 98.2%, respectively) for a week after disinfection.
Identification of
Ruminococcus flavefaciens
as the Predominant Cellulolytic Bacterial Species of the Equine Cecum
Authors: Julliand Veronique , de Vaux Albane , Millet Liliane , Fonty Gerard
Published: December 2019

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that
Ruminococcus flavefaciens
was the predominant species in the pony and donkey cecum.
Fibrobacter succinogenes
and
Ruminococcus albus
were present at low levels. Four isolates, morphologically resembling
R. flavefaciens
, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.
A Nisin Bioassay Based on Bioluminescence
Authors: Wahlström G. , Saris P. E. J.
Published: December 2019

A
Lactococcus lactis
subsp.
lactis
strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods.
Polycyclic Aromatic Hydrocarbon Metabolism by White Rot Fungi and Oxidation by
Coriolopsis gallica
UAMH 8260 Laccase
Authors: Pickard Michael A. , Roman Rosa , Tinoco Raunel , Vazquez-Duhalt Rafael
Published: December 2019

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 μg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from
Coriolopsis gallica
. The reaction mixtures contained 20 μM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[
a
]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (
k
) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (
k
= 2.4 h
−1
) that ABTS (1 mM) supported (
k
= 5.2 h
−1
), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h
−1
. Laccase purified from
Pleurotus ostreatus
had an activity similar to that of
C. gallica
laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present.
Spatial Heterogeneity of Bacterial Populations along an Environmental Gradient at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)
Authors: Sievert Stefan M. , Brinkhoff Thorsten , Muyzer Gerard , Ziebis Wiebke , Kuever Jan
Published: December 2019

The spatial heterogeneity of bacterial populations at a shallow-water hydrothermal vent in the Aegean Sea close to the island of Milos (Greece) was examined at two different times by using acridine orange staining for total cell counts, cultivation-based techniques, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. Concurrent with measurements of geochemical parameters, samples were taken along a transect from the center of the vent to the surrounding area. Most-probable-number (MPN) counts of metabolically defined subpopulations generally constituted a minor fraction of the total cell counts; both counting procedures revealed the highest cell numbers in a transition zone from the strongly hydrothermally influenced sediments to normal sedimentary conditions. Total cell counts ranged from 3.2 × 10
5
cells ml
−1
in the water overlying the sediments to 6.4 × 10
8
cells g (wet weight) of sediment
−1
. MPN counts of chemolithoautotrophic sulfur-oxidizing bacteria varied between undetectable and 1.4 × 10
6
cells g
−1
. MPN counts for sulfate-reducing bacteria and dissimilatory iron-reducing bacteria ranged from 8 to 1.4 × 10
5
cells g
−1
and from undetectable to 1.4 × 10
6
cells g
−1
, respectively. DGGE revealed a trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone to a community dominated by few populations close to the center of the vent. Temperature was found to be an important parameter in determining this trend. However, at one sampling time this trend was not discernible, possibly due to storm-induced disturbance of the upper sediment layers.
Transduction of Enteric
Escherichia coli
Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from
Escherichia coli
O157:H7
Authors: Schmidt Herbert , Bielaszewska Martina , Karch Helge
Published: December 2019

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric
Escherichia coli
strains and to develop infectious progeny from such lysogenized strains. The

stx
2

gene of the patient
E. coli
O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (
cat
) gene from plasmid pACYC184. Phage φ3538(Δ

stx
2

::
cat
) was isolated after induction of
E. coli
O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic
E. coli
(EPEC), enteroinvasive
E. coli
(EIEC), Stx-producing
E. coli
(STEC), enterotoxigenic
E. coli
(ETEC), enteroaggregative
E. coli
(EAEC), and
E. coli
from the physiological stool microflora were infected with φ3538(Δ

stx
2

::
cat
), and plaque formation and lysogenic conversion of wild-type
E. coli
strains were investigated. With the exception of one EIEC strain, none of the
E. coli
strains supported the formation of plaques when used as indicators for φ3538(Δ

stx
2

::
cat
). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6
E. coli
isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of φ3538(Δ

stx
2

::
cat
) that formed plaques on a lawn of
E. coli
laboratory strain C600. The results of our study demonstrate that φ3538(Δ

stx
2

::
cat
) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic
E. coli
and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread
stx
genes among enteric
E. coli
strains.
Identification of an NADH-Cytochrome b
5
Reductase Gene from an Arachidonic Acid-Producing Fungus,
Mortierella alpina
1S-4, by Sequencing of the Encoding cDNA and Heterologous Expression in a Fungus,
Aspergillus oryzae
Authors: Sakuradani Eiji , Kobayashi Michihiko , Shimizu Sakayu
Published: December 2019

Based on the sequence information for bovine and yeast NADH-cytochrome
b
5
reductases (CbRs), a DNA fragment was cloned from
Mortierella alpina
1S-4 after PCR amplification. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (
Saccharomyces cerevisiae
), bovine, human, and rat CbRs. These results suggested that this cDNA is a CbR gene. The results of a structural comparison of the flavin-binding β-barrel domains of CbRs from various species and that of the
M. alpina
enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds. The corresponding genomic gene, which was also cloned from
M. alpina
1S-4 by means of a hybridization method with the above probe, had four introns of different sizes. These introns had GT at the 5′ end and AG at the 3′ end, according to a general GT-AG rule. The expression of the full-length cDNA in a filamentous fungus,
Aspergillus oryzae
, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes. The
M. alpina
CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity. The purified CbR preferred NADH over NADPH as an electron donor. This is the first report of an analysis of this enzyme in filamentous fungi.