bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of an Acetyl Xylan Esterase fromBacillus pumilus
Authors: Degrassi Giuliano , Okeke Benedict C. , Bruschi Carlo V. , Venturi Vittorio
Published: December 2019

Bacillus pumilusPS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km) andVmaxfor α-naphthyl acetate were 1.54 mM and 360 μmol min−1mg of protein−1, respectively.
Mercury Methylation and Demethylation in Anoxic Lake Sediments and by Strictly Anaerobic Bacteria
Authors: Pak K.-R. , Bartha R.
Published: December 2019

After spiking anoxic sediment slurries of three acidic oligotrophic lakes with either HgCl
2
at 1.0 μg/ml or CH
3
HgI at 0.1 μg/ml, both mercury methylation and demethylation rates were measured. High mercury methylation potentials were accompanied by high demethylation potentials in the same sediment. These high potentials correlated positively with the concentrations of organic matter and dissolved sulfate in the sediment and with mercury levels in fish. Adjustment of the acidic sediment pH to neutrality failed to influence either the methylation or the demethylation rate of mercury. The opposing methylation and demethylation processes converged to establish similar Hg
2+
-CH
3
Hg
+
equilibria in all three sediments. Because of their metabolic dominance in anoxic sediments, mercury methylation and demethylation in pure cultures of sulfidogenic, methanogenic, and acetogenic bacteria were also measured. Sulfidogens both methylated and demethylated mercury, but the methanogen tested only catalyzed demethylation and the acetogen neither methylated nor demethylated mercury.
A Fermentor System for Regulating Oxygen at Low Concentrations in Cultures of
Saccharomyces cerevisiae
Authors: Burke Patricia V. , Kwast Kurt E. , Everts Frank , Poyton Robert O.
Published: December 2019

The growth of yeast cells to high densities at low, but constant, oxygen concentrations is difficult because the cells themselves respire oxygen; hence, as cell mass increases, so does oxygen consumption. To circumvent this problem, we have designed a system consisting of a computer-controlled gas flow train that adjusts oxygen concentration in the gas flow to match cellular demand. It does this by using a proportional-integral-differential algorithm in conjunction with a three-way valve to mix two gases, adjusting their proportions to maintain the desired oxygen concentration. By modeling yeast cell yields at intermediate to low oxygen concentrations, we have found that cellular respiration declines with oxygen concentration, most likely because of a decrease in the expression of genes for respiratory proteins. These lowered rates of oxygen consumption, together with the gas flow system described here, allow the growth of yeast cells to high densities at low oxygen concentrations. This system can also be used to grow cells at any desired oxygen concentration and for regulated shifts between oxygen concentrations.
Ultradian Growth in
Prochlorococcus
spp
Authors: Shalapyonok Alexi , Olson Robert J. , Shalapyonok Ludmila S.
Published: December 2019

Species of the widespread marine prokaryote
Prochlorococcus
exhibited ultradian growth (faster than 1 division per day) both in situ and in culture, even though cell division is strictly phased to the light-dark cycle. Under optimal conditions a second DNA replication and cell division closely followed, but did not overlap with, the first division. The timing of cell cycle events was not affected by light intensity or duration, suggesting control by a light-triggered timer or circadian clock rather than by completion of a light-dependent assimilation phase. This mode of ultradian growth has not been observed previously and poses new questions about the regulation of cellular rhythms in prokaryotes. In addition, it implies that conclusions regarding the lack of nutrient limitation of
Prochlorococcus
in the open ocean, which were based on the appearance that cells were growing at their maximal rate, need to be reconsidered.
Methanol Improves Methane Uptake in Starved Methanotrophic Microorganisms
Authors: Jensen Sigmund , Priemé Anders , Bakken Lars
Published: December 2019

Methanotrophs in enrichment cultures grew and sustained atmospheric methane oxidation when supplied with methanol. If they were not supplied with methanol or formate, their atmospheric methane oxidation came to a halt, but it was restored within hours in response to methanol or formate. Indigenous forest soil methanotrophs were also dependent on a supply of methanol upon reduced methane access but only when exposed to a methane-free atmosphere. Their immediate response to each methanol addition, however, was to shut down the oxidation of atmospheric methane and to reactivate atmospheric methane oxidation as the methanol was depleted.
Lipopolysaccharide Profiles from Nodules as Markers of
Bradyrhizobium
Strains Nodulating Wild Legumes
Authors: Santamaría Mónica , Gutiérrez-Navarro Ángel M. , Corzo Javier
Published: December 2019

To develop the use of electrophoretic lipopolysaccharide profiles for
Bradyrhizobium
strain identification, we studied the feasibility of using electrophoresis of whole legume nodule homogenates to obtain distinctive lipopolysaccharide profiles. The electrophoretic patterns were the same whether we used nodule extracts, bacteroids, or cultured bacteria as samples, and there was no evidence of changes in the ladder-like pattern during the nodulation process. To assess the reliability of using lipopolysaccharide profiling performed with individual nodules for studying the diversity and microdistribution of the rhizobia nodulating wild shrub legumes, we used a population of
Adenocarpus foliolosus
seedlings. We obtained 75 different profiles from the 147 nodules studied. There was no dominant profile in the sample, and a plant with different nodules generally produced different profiles. Electrophoresis of legume root nodules proved to be a fast and discriminating technique for determining the diversity of a bradyrhizobial population, although it did not allow the genetic relationships among the nodulating strains to be studied.
Complete Reductive Dehalogenation of Brominated Biphenyls by Anaerobic Microorganisms in Sediment
Authors: Bedard Donna L. , Van Dort Heidi M.
Published: December 2019

We sought to determine whether microorganisms from the polychlorinated biphenyl (PCB)-contaminated sediment in Woods Pond (Lenox, Mass.) could dehalogenate brominated biphenyls. The PCB dechlorination specificities for the microorganisms in this sediment have been well characterized. This allowed us to compare the dehalogenation specificities for brominated biphenyls and chlorinated biphenyls within a single sediment. Anaerobic sediment microcosms were incubated separately at 25°C with 16 different mono- to tetrabrominated biphenyls (350 μM) and disodium malate (10 mM). Samples were extracted and analyzed by gas chromatography with an electron capture detector and a mass spectrometer detector at various times for up to 54 weeks. All of the tested brominated biphenyls were dehalogenated. For most congeners, including 2,6-dibromobiphenyl (26-BB) and 24-25-BB, the dehalogenation began within 1 to 2 weeks. However, for 246-BB and 2-2-BB, debromination was first observed at 7 and 14 weeks, respectively. Most intermediate products did not persist, but when 2-2-BB was produced as a dehalogenation product, it persisted for at least 15 weeks before it was dehalogenated to 2-BB and then to biphenyl. The dehalogenation specificities for brominated and chlorinated biphenyls were similar:
meta
and
para
substituents were generally removed first, and
ortho
substituents were more recalcitrant. However, the brominated biphenyls were better dehalogenation substrates than the chlorinated biphenyls. All of the tested bromobiphenyls, including those with
ortho
and unflanked
meta
and
para
substituents, were ultimately dehalogenated to biphenyl, whereas their chlorinated counterparts either were not dehalogenation substrates or were only partially dehalogenated. Our data suggest that PCB-dechlorinating microorganisms may be able to dehalogenate brominated biphenyls and may exhibit a relaxed specificity for these substrates.
Distribution of a Population ofRhizobium leguminosarumbv. trifolii among Different Size Classes of Soil Aggregates
Authors: Mendes Ieda C. , Bottomley Peter J.
Published: December 2019

A combination of the plant infection-soil dilution technique (most-probable-number [MPN] technique) and immunofluorescence direct count (IFDC) microscopy was used to examine the effects of three winter cover crop treatments on the distribution of a soil population ofRhizobium leguminosarumbv. trifolii across different size classes of soil aggregates (<0.25, 0.25 to 0.5, 0.5 to 1.0, 1.0 to 2.0, and 2.0 to 5.0 mm). The aggregates were prepared from a Willamette silt loam soil immediately after harvest of broccoli (September 1995) and before planting and after harvest of sweet corn (June and September 1996, respectively). The summer crops were grown in soil that had been either fallowed or planted with a cover crop of red clover (legume) or triticale (cereal) from September to April. TheRhizobiumsoil population was heterogeneously distributed across the different size classes of soil aggregates, and the distribution was influenced by cover crop treatment and sampling time. On both September samplings, the smallest size class of aggregates (<0.25 mm) recovered from the red clover plots carried between 30 and 70% of the total nodulatingR. leguminosarumpopulation, as estimated by the MPN procedure, while the same aggregate size class from the June sampling carried only ∼6% of the population. In June, IDFC microscopy revealed that the 1.0- to 2.0-mm size class of aggregates from the red clover treatment carried a significantly greater population density of the successful nodule-occupying serotype, AR18, than did the aggregate size classes of <0.5 mm, and 2 to 5 mm. In September, however, the population profile of AR18 had shifted such that the density was significantly greater in the 0.25- to 0.5-mm size class than in aggregates of <0.25 mm and >1.0 mm. The populations of two otherRhizobiumserotypes (AR6 and AS36) followed the same trends of distribution in the June and September samplings. These data indicate the existence of structural microsites that vary in their suitabilities to support growth and protection of bacteria and that are influenced by the presence and type of plant grown in the soil.
Population Dynamics of Phenol-Degrading Bacteria in Activated Sludge Determined by
gyrB
-Targeted Quantitative PCR
Authors: Watanabe Kazuya , Yamamoto Satoshi , Hino Sanae , Harayama Shigeaki
Published: December 2019

A method for quantifying bacterial populations introduced into an activated-sludge microbial community is described. The method involves extraction of DNA from activated sludge, appropriate dilution of the extracted DNA with DNA extracted from nonintroduced activated sludge, PCR amplification of a
gyrB
gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. The adequacy of the method was examined by analyzing the population dynamics of two phenol-degrading bacteria,
Pseudomonas putida
BH and
Comamonas
sp. strain E6, that had been introduced into phenol-digesting activated sludge. The density of each of the two populations determined by the PCR method immediately after the introduction was consistent with the density estimated from a plate count of the inoculum. This quantitative PCR method revealed different population dynamics for the two strains in the activated sludge under different phenol-loading conditions. The behavior of both of these strains in the activated sludge reflected the growth kinetics of the strains determined in laboratory axenic cultures.
Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition
Authors: Nüsslein Klaus , Tiedje James M.
Published: December 2019

The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of thePseudomonas,Rhizobium-Agrobacterium, andRhodospirillumassemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with severalClostridiumassemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community.
Buffering Capacity and Membrane H
+
Conductance of Neutrophilic and Alkalophilic Gram-Positive Bacteria
Authors: Rius Núria , Lorén José G.
Published: December 2019

Buffering capacity and membrane H
+
conductance were examined in three gram-positive bacteria,
Staphylococcus aureus
,
Bacillus subtilis
, and
Bacillus alcalophilus
. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H
+
conductance of
B. alcalophilus
are influenced by the pH of the medium and the culture conditions. Suspensions of
B. alcalophilus
cells from both H. A. medium and
l
-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5.
B. alcalophilus
grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria
S. aureus
and
B. subtilis
. Fermenting cells exhibited significantly higher values for both variables than respiring cells.
COVASIAM: an Image Analysis Method That Allows Detection of Confluent Microbial Colonies and Colonies of Various Sizes for Automated Counting
Authors: Corkidi G. , Diaz-Uribe R. , Folch-Mallol J. L. , Nieto-Sotelo J.
Published: December 2019

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties:
Saccharomyces cerevisiae
,
Aspergillus nidulans
,
Escherichia coli
,
Azotobacter vinelandii
,
Pseudomonas aeruginosa
, and
Rhizobium etli
. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (ς = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (ς = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.
Structural and Kinetic Properties of Nonglycosylated RecombinantPenicillium amagasakienseGlucose Oxidase Expressed inEscherichia coli
Authors: Witt Susanne , Singh Mahavir , Kalisz Henryk M.
Published: December 2019

The gene coding forPenicillium amagasakienseglucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. RecombinantEscherichia coliexpression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed intoE. coliTG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml ofE. coliculture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml−1in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-literE. coliculture. Circular dichroism demonstrated similar conformations for recombinant and nativeP. amagasakienseGOXs. The purified enzyme has a specific activity of 968 U mg−1and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX fromP. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX inE. coli.
Failure To Differentiate
Cryptosporidium parvum
from
C. meleagridis
Based on PCR Amplification of Eight DNA Sequences
Authors: Champliaud Dominique , Gobet Philippe , Naciri Muriel , Vagner Odile , Lopez José , Buisson Jean Christophe , Varga Istvan , Harly Géraldine , Mancassola Roseline , Bonnin Alain
Published: December 2019

In order to determine the specificities of PCR-based assays used for detecting
Cryptosporidium parvum
DNA, eight pairs of previously described PCR primers targeting six distinct regions of the
Cryptosporidium
genome were evaluated for the detection of
C. parvum
, the agent of human cryptosporidiosis, and
C. muris
,
C. baileyi
, and
C. meleagridis
, three
Cryptosporidium
species that infect birds or mammals but are not considered to be human pathogens. The four
Cryptosporidium
species were divided into two groups:
C. parvum
and
C. meleagridis
, which gave the same-sized fragments with all the reactions, and
C. muris
and
C. baileyi
, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification,
C. parvum
and
C. meleagridis
couldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen
C. parvum.
Release of Dimethylsulfide from Dimethylsulfoniopropionate by Plant-Associated Salt Marsh Fungi
Authors: Bacic M. K. , Newell S. Y. , Yoch D. C.
Published: December 2019

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus
Fusarium
[M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106–111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera
Halophytophthora
and
Pythium
, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer’s ability to catabolize DMSP via the DMSP lyase pathway and the host plant’s production of DMSP as a secondary product.
Extremely Barophilic Bacteria Isolated from the Mariana Trench, Challenger Deep, at a Depth of 11,000 Meters
Authors: Kato Chiaki , Li Lina , Nogi Yuichi , Nakamura Yuka , Tamaoka Jin , Horikoshi Koki
Published: December 2019

Two strains of obligately barophilic bacteria were isolated from a sample of the world’s deepest sediment, which was obtained by the unmanned deep-sea submersible
Kaiko
in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to
Shewanella benthica
and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus
Moritella
. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus
Moritella
isolated from the deep-sea environment.
Analysis of Stable Low-Molecular-Weight RNA Profiles of Members of the Family
Rhizobiaceae
Authors: Velázquez Encarna , Cruz-Sánchez JoséMaría , Mateos Pedro F. , Martínez-Molina Eustoquio
Published: December 2019

Staircase electrophoresis in polyacrylamide gels was used to analyze the stable low-molecular-weight (LMW) RNA profiles of 24 type strains belonging to the family
Rhizobiaceae
. This new electrophoretic technique results in good separation of the molecules forming the LMW RNA profiles. Differences in the number and distribution of the RNA bands in these profiles allowed us to identify differences among the 24 strains assayed. Species assignments based on LMW RNAs proved to be consistent with the established taxonomic classification. Analysis of the data obtained and the corresponding dendrograms revealed relationships between genera and species; these relationships were essentially the same as those obtained with other techniques, such as DNA hybridization and 16S rRNA sequencing. Use of the technique described here, with which it is possible to analyze a large number of strains in a short time, permits rapid identification of species belonging to the family
Rhizobiaceae
and should in the future facilitate biodiversity studies and detection of new species.
Influence of Cold Stress on the Preliminary Enrichment Time Needed for Detection of Enterohemorrhagic
Escherichia coli
in Ground Beef by PCR
Authors: Uyttendaele M. , Grangette C. , Rogerie F. , Pasteau S. , Debevere J. , Lange M.
Published: December 2019

The influence of cold stress at 4 and 0°C on the detection time as assessed by impedance technology (Bactometer; Biomérieux, Marcy l’Etoile, France) of different enterohemorrhagic
Escherichia coli
(EHEC) strains was determined. Although there is some variation in susceptibility among EHEC strains, prolonged exposure of EHEC to cold stress, i.e., 4 and 5 days at 4 and 0°C, respectively, in general significantly increased their detection time. This reflects an increase of the lag-phase time caused by cold stress. Two EHEC strains were selected to determine the minimum preliminary enrichment time that would ensure a positive PCR detection of low numbers of verotoxin-producing
E. coli
(VTEC; 2 to 2 × 10
5
CFU/25 g) inoculated into ground beef (25 g) and stored at 4 or −20°C for 8 and 14 days, respectively. Incubation times of 6 and 9 h of 1 to 10 CFU/g and 1 to 10 CFU/25 g, respectively, were sufficient for PCR detection of VTEC in ground beef when analysis was performed immediately after inoculation (no cold stress). When cells are exposed to cold stress (4 or −20°C) a 24-h enrichment period is recommended. Restriction of enrichment time to 9 h under these circumstances decreases the sensitivity of PCR detection to 80 CFU/g. Hence, to obtain maximum sensitivity, PCR detection of VTEC in naturally contaminated ground beef should be performed after 24 h of enrichment.
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

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

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

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

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

A 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

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

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

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

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

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

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

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

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
.