bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of an Amoeba Naturally Harboring a Distinctive
Legionella
Species
Authors: Newsome Anthony L. , Scott Tammy M. , Benson Robert F. , Fields Barry S. 
Published: December 2019

ABSTRACT

There are numerous in vitro studies documenting the multiplication of
Legionella
species in free-living amoebae and other protozoa. It is believed that protozoa serve as host cells for the intracellular replication of certain
Legionella
species in a variety of environmental settings. This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample. In the laboratory, the bacterium multiplied within and was highly pathogenic for
Acanthamoeba polyphaga
. Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media. A 16S rRNA gene analysis placed the bacterium within the genus
Legionella
. Serological studies indicate that it is distinct from previously described species of the genus. This report also describes methods that should prove useful for the isolation and characterization of additional
Legionella
-like bacteria from free-living amoebae. In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens.
Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, in
Escherichia coli
Authors: Nishihara Kazuyo , Kanemori Masaaki , Kitagawa Masanari , Yanagi Hideki , Yura Takashi 
Published: December 2019

ABSTRACT

Plasmids that can be used for controlled expression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly of recombinant proteins in
Escherichia coli
. A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when
l
-arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable when it was produced in
E. coli
K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor ς
32
revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of ς
32
at certain temperatures.
Modelling the Growth Limits (Growth/No Growth Interface) of
Escherichia coli
as a Function of Temperature, pH, Lactic Acid Concentration, and Water Activity
Authors: Presser K. A. , Ross T. , Ratkowsky D. A. 
Published: December 2019

ABSTRACT

The form of a previously developed Bělehrádek type of growth rate model was used to develop a probability model for defining the growth/no growth interface as a function of temperature (10 to 37°C), pH (pH 2.8 to 6.9), lactic acid concentration (0 to 500 mM), and water activity (0.955 to 0.999; NaCl was used as the humectant).
Escherichia coli
was unable to grow in broth in which the undissociated lactic acid concentration exceeded 11 mM or, with two exceptions, at a pH of 3.9 or less with no lactic acid present. Under experimental conditions at which the pH and the undissociated acid concentrations were the major growth-limiting factors, the growth/no growth interface was essentially independent of temperature at temperatures ranging from 15 to 37°C. The interface between conditions that allowed growth and conditions at which growth did not occur was abrupt. The inhibitory effect of combinations of water activity and pH varied with temperature. Predictions of the model for the growth/no growth interface were consistent with 95% of the experimental data set.
Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance
Authors: Rohr Ute , Weber Susanne , Michel Rolf , Selenka Fidelis , Wilhelm Michael 
Published: December 2019

ABSTRACT

Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as
Hartmannella vermiformis
(65%),
Echinamoebae
spp. (15%),
Saccamoebae
spp. (12%), and
Vahlkampfia
spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified:
Acanthamoeba
(22%),
Naegleria
(22%),
Vahlkampfia
(20%),
Hartmannella
(15%), and
Vanella
(7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six
Acanthamoeba
isolates from the moist areas were considered potential pathogens. Four
Hartmannella
and two
Saccamoeba
isolates from hot water could be cultured at 53°C.
Cloning and Characterization of a Prolinase Gene (pepR) fromLactobacillus rhamnosus
Authors: Varmanen Pekka , Rantanen Terhi , Palva Airi , Tynkkynen Soile 
Published: December 2019

ABSTRACTA peptidase gene expressingl-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library ofLactobacillus rhamnosus1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kbSacI fragment. A sequence analysis of theSacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein fromLactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) followingpepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated thatpepRis expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain ofL. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu inL. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The clonedpepRexpressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substratesl-leucine-β-naphthylamide andl-phenylalanine-β-naphthylamide were hydrolyzed by the PepR ofL. rhamnosus.
Genetically Engineered
Saccharomyces
Yeast Capable of Effective Cofermentation of Glucose and Xylose
Authors: Ho Nancy W. Y. , Chen Zhengdao , Brainard Adam P. 
Published: December 2019

ABSTRACT

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However,
Saccharomyces
spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transform
Saccharomyces
spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2μm-based high-copy-number yeast-
E. coli
shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from
Pichia stipitis
), and a xylulokinase gene (from
Saccharomyces cerevisiae
). These xylose-metabolizing genes were also fused to signals controlling gene expression from
S. cerevisiae
glycolytic genes. Transformation of
Saccharomyces
sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.
Rapid Consumption of Low Concentrations of Methyl Bromide by Soil Bacteria
Authors: Hines Mark E. , Crill Patrick M. , Varner Ruth K. , Talbot Robert W. , Shorter Joanne H. , Kolb Charles E. , Harriss Robert C. 
Published: December 2019

ABSTRACT

A dynamic dilution system for producing low mixing ratios of methyl bromide (MeBr) and a sensitive analytical technique were used to study the uptake of MeBr by various soils. MeBr was removed within minutes from vials incubated with soils and ∼10 parts per billion by volume of MeBr. Killed controls did not consume MeBr, and a mixture of the broad-spectrum antibiotics chloramphenicol and tetracycline inhibited MeBr uptake by 98%, indicating that all of the uptake of MeBr was biological and by bacteria. Temperature optima for MeBr uptake suggested a biological sink, yet soil moisture and temperature optima varied for different soils, implying that MeBr consumption activity by soil bacteria is diverse. The eucaryotic antibiotic cycloheximide had no effect on MeBr uptake, indicating that soil fungi were not involved in MeBr removal. MeBr consumption did not occur anaerobically. A dynamic flowthrough vial system was used to incubate soils at MeBr mixing ratios as low as those found in the remote atmosphere (5 to 15 parts per trillion by volume [pptv]). Soils consumed MeBr at all mixing ratios tested. Temperate forest and grassy lawn soils consumed MeBr most rapidly (rate constant [
k
] = 0.5 min
−1
), yet sandy temperate, boreal, and tropical forest soils also readily consumed MeBr. Amendments of CH
4
up to 5% had no effect on MeBr uptake even at CH
4
:MeBr ratios of 10
7
, and depth profiles of MeBr and CH
4
consumption exhibited very different vertical rate optima, suggesting that methanotrophic bacteria, like those presently in culture, do not utilize MeBr when it is at atmospheric mixing ratios. Data acquired with gas flux chambers in the field demonstrated the very rapid in situ consumption of MeBr by soils. Uptake of MeBr at mixing ratios found in the remote atmosphere occurs via aerobic bacterial activity, displays first-order kinetics at mixing ratios from 5 pptv to ∼1 part per million per volume, and is rapid enough to account for 25% of the global annual loss of atmospheric MeBr.
Effect of Bacterial Distribution and Activity on Conjugal Gene Transfer on the Phylloplane of the Bush Bean (
Phaseolus vulgaris
)
Authors: Normander Bo , Christensen Bjarke B. , Molin Søren , Kroer Niels 
Published: December 2019

ABSTRACT

Conjugal plasmid transfer was examined on the phylloplane of bean (
Phaseolus vulgaris
) and related to the spatial distribution pattern and metabolic activity of the bacteria. The donor (
Pseudomonas putida
KT2442) harbored a derivative of the TOL plasmid, which conferred kanamycin resistance and had the
gfp
gene inserted downstream of a
lac
promoter. A chromosomal insertion of
lacI
q
prevented expression of the
gfp
gene. The recipient (
P. putida
KT2440) had a chromosomal tetracycline resistance marker. Thus, transconjugants could be enumerated by plating and visualized in situ as green fluorescent cells. Sterile bean seedlings were inoculated with donors and recipients at densities of approximately 10
5
cells per cm
2
. To manipulate the density and metabolic activity (measured by incorporation of [
3
H]leucine) of the inoculated bacteria, plants were grown at various relative humidities (RH). At 100% RH, the transconjugants reached a density of 3 × 10
3
CFU/cm
2
, corresponding to about one-third of the recipient population. At 25% RH, numbers of transconjugants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after 96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.
Evaluation of Structurally Different Carotenoids in
Escherichia coli
Transformants as Protectants against UV-B Radiation
Authors: Sandmann Gerhard , Kuhn Silvia , Böger Peter 
Published: December 2019

ABSTRACT

Escherichia coli
cells transformed with several carotenogenic genes to mediate the formation of ζ-carotene, neurosporene, lycopene, β-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and β-carotene protected
E. coli
against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.
Effects of Above-Optimum Growth Temperature and Cell Morphology on Thermotolerance of
Listeria monocytogenes
Cells Suspended in Bovine Milk
Authors: Rowan Neil J. , Anderson John G. 
Published: December 2019

ABSTRACT

The thermotolerances of two different cell forms of
Listeria monocytogenes
(serotype 4b) grown at 37 and 42.8°C in commercially pasteurized and laboratory-tyndallized whole milk (WM) were investigated. Test strains, after growth at 37 or 42.8°C, were suspended in WM at concentrations of approximately 1.5 × 10
8
to 3.0 × 10
8
cells/ml and were then heated at 56, 60, and 63°C for various exposure times. Survival was determined by enumeration on tryptone-soya-yeast extract agar and
Listeria
selective agar, and D values (decimal reduction times) and Z values (numbers of degrees Celsius required to cause a 10-fold change in the D value) were calculated. Higher average recovery and higher D values (i.e., seen as a 2.5- to 3-fold increase in thermotolerance) were obtained when cells were grown at 42.8°C prior to heat treatment. A relationship was observed between thermotolerance and cell morphology of
L. monocytogenes
. Atypical
Listeria
cell types (consisting predominantly of long cell chains measuring up to 60 μm in length) associated with rough (R) culture variants were shown to be 1.2-fold more thermotolerant than the typical dispersed cell form associated with normal smooth (S) cultures (
P
≤ 0.001). The thermal death-time (TDT) curves of R-cell forms contained a tail section in addition to the shoulder section characteristic of TDT curves of normal single to paired cells (i.e., S form). The factors shown to influence the thermoresistance of suspended
Listeria
cells (
P
≤ 0.001) were as follows: growth and heating temperatures, type of plating medium, recovery method, and cell morphology. Regression analysis of nonlinear data can underestimate survival of
L. monocytogenes
; the end point recovery method was shown to be a better method for determining thermotolerance because it takes both shoulders and tails into consideration. Despite their enhanced heat resistance, atypical R-cell forms of
L. monocytogenes
were unable to survive the low-temperature, long-time pasteurization process when freely suspended and heated in WM.
Construction of an Efficient Biologically Contained
Pseudomonas putida
Strain and Its Survival in Outdoor Assays
Authors: Molina Lázaro , Ramos Cayo , Ronchel María-Carmen , Molin Søren , Ramos Juan L. 
Published: December 2019

ABSTRACT

Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn
5
transposon bearing a fusion of the P

lac

promoter to the
gef
killing gene and a fusion of the Pm promoter to the
lacI
gene plus the positive regulator of the Pm promoter, the
xylS
gene. This mini-Tn
5
transposon was transferred to the chromosome of
Pseudomonas putida
CMC4, and in culture this strain survived in the presence of 3-methylbenzoate (an XylS effector) and committed suicide in the absence of this aromatic compound. The rate of killing escape was on the order of 10
−8
per cell and per generation. This contained strain and an uncontained control strain were used in outdoor tests performed in the spring-summer and autumn-winter periods to determine their survival in planted and unplanted soils with and without 3-methylbenzoate. In unplanted soils the numbers of both the contained strain and the uncontained strain per gram of soil tended to decrease, but the numbers of the contained strain decreased faster in soils without 3-methylbenzoate. The decrease in the number of CFU per gram of soil was faster in the spring-summer period than in the autumn-winter period. In planted soils survival in the rhizosphere and survival in bulk soil were studied. In the rhizosphere the uncontained control strain tended to become established at levels on the order of 10
5
to 10
6
CFU/g of soil regardless of the presence of 3-methylbenzoate. In the bulk soil the numbers of bacterial cells were 2 to 3 orders of magnitude lower. In planted soils the contained strain tended to disappear, but this tendency was more pronounced in the absence of 3-methylbenzoate and occurred faster in the summer assay than in the winter assay. We found no evidence of dispersal of the test strains outside the experimental plots.
Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from
Bacillus subtilis
Authors: Kerovuo Janne , Lauraeus Marko , Nurminen Päivi , Kalkkinen Nisse , Apajalahti Juha 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (
phyC
) was cloned from the
B. subtilis
VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.
Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in
Burkholderia cepacia
AC1100: Characterization of the
tftC
and
tftD
Genes and Locations of the
tft
Operons on Multiple Replicons
Authors: Hübner Anette , Danganan Clyde E. , Xun Luying , Chakrabarty A. M. , Hendrickson William 
Published: December 2019

ABSTRACT

Burkholderia cepacia
AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the
tftC
and
tftD
genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the
tftC
and
tftD
genes from a genomic library of AC1100. The derived amino acid sequences of
tftC
and
tftD
show significant homology to the two-component monooxygenases HadA of
Burkholderia pickettii
, HpaBC of
Escherichia coli
, and HpaAH of
Klebsiella pneumonia
. Expression of the
tftC
and
tftD
genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned
tft
genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of
B. cepacia
AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The
tft
genes are located on the smaller replicons: the
tftAB
cluster is on replicon IV,
tftEFGH
is on replicon III, and copies of the
tftC
and the
tftCD
operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the
tftA
and
tftB
genes translocated from one replicon to another, with the concomitant loss of
tftEFGH
and one copy of
tftCD
.
Estimation of Nitrifying Bacterial Activities by Measuring Oxygen Uptake in the Presence of the Metabolic Inhibitors Allylthiourea and Azide
Authors: Ginestet Philippe , Audic Jean-Marc , Urbain Vincent , Block Jean-Claude 
Published: December 2019

ABSTRACT
The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 μM) and azide (24 μM) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.
Seasonal Abundance of Lysogenic Bacteria in a Subtropical Estuary
Authors: Cochran Pamela K. , Paul John H. 
Published: December 2019

ABSTRACT
Seasonal changes in the abundance of inducible lysogenic bacteria in a eutrophic estuarine environment were investigated over a 13-month period. Biweekly water samples were collected from Tampa Bay, Fla., and examined for prophage induction by mitomycin C treatment. At the conclusion of the study, we determined that 52.2% of the samples displayed prophage induction, as indicated by significant increases in viral direct counts compared with uninduced controls. Samples that displayed prophage induction occurred during the warmer months (February through October), when surface water temperatures were above 19°C, and no induction was observed in November, December, or January. This study presents clear evidence that there is seasonal variation in the number of inducible lysogenic bacteria in an estuarine environment.