bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a PCR Assay for Detection of
Yersinia ruckeri
in Tissues of Inoculated and Naturally Infected Trout
Authors: Gibello A. , Blanco M. M. , Moreno M. A. , Cutuli M. T. , Domenech A. , Domínguez L. , Fernández-Garayzábal J. F.
Published: December 2019

A PCR-based method was developed for the specific detection of
Yersinia ruckeri
in tissues of inoculated trout and naturally infected trout. No amplification products were obtained with other yersiniae, bacterial fish pathogens, or phylogenetically related bacteria (
n
= 34). The sensitivity of PCR detection was 60 to 65 bacterial cells per PCR tube, which was decreased to 10 to 20 cells by hybridization with a nonradioactive probe. The PCR assay proved to be as reliable as and faster than the conventional culture method for the detection of
Y. ruckeri
in infected trout tissues.
Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source
Authors: Coulter Catherine , Hamilton John T. G. , McRoberts W. Colin , Kulakov Leonid , Larkin Michael J. , Harper David B.
Published: December 2019

A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH3Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH3Cl; then it catalyzed methyl transfer from CH3Cl, CH3Br, or CH3I to the following acceptor ions (in order of decreasing efficacy): I−, HS−, Cl−, Br−, NO2−, CN−, and SCN−. Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase fromEscherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N2O, and Hg2+to affect the methyltransferase suggests significant differences. During CH3Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS−, a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters withRhizobiumspp. in the alpha subdivision of theProteobacteriaand is closely related to strain IMB-1, a recently isolated CH3Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899–2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.
Nitrogen Availability to
Pseudomonas fluorescens
DF57 Is Limited during Decomposition of Barley Straw in Bulk Soil and in the Barley Rhizosphere
Authors: Jensen Linda Elise , Nybroe Ole
Published: December 2019

The availability of nitrogen to
Pseudomonas fluorescens
DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism
Trichoderma harzianum
Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in
Pseudomonas
is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities.
Simultaneous Reduction of Nitrate and Selenate by Cell Suspensions of Selenium-Respiring Bacteria
Authors: Oremland Ronald S. , Blum Jodi Switzer , Bindi Allana Burns , Dowdle Philip R. , Herbel Mitchell , Stolz John F.
Published: December 2019

Washed-cell suspensions ofSulfurospirillum barnesiireduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ∼11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphileBacillus arsenicoselenatisgave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted forBacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations ofS. barnesiisuggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with75Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.
Identification of Phytoplankton from Flow Cytometry Data by Using Radial Basis Function Neural Networks
Authors: Wilkins M. F. , Boddy Lynne , Morris C. W. , Jonker R. R.
Published: December 2019

We describe here the application of a type of artificial neural network, the Gaussian radial basis function (RBF) network, in the identification of a large number of phytoplankton strains from their 11-dimensional flow cytometric characteristics measured by the European Optical Plankton Analyser instrument. The effect of network parameters on optimization is examined. Optimized RBF networks recognized 34 species of marine and freshwater phytoplankton with 91.5% success overall. The relative importance of each measured parameter in discriminating these data and the behavior of RBF networks in response to data from “novel” species (species not present in the training data) were analyzed.
Pelagic-Benthic Coupling of Nucleic Acids in an Abyssal Location of the Northeastern Atlantic Ocean
Authors: Dell’Anno A. , Fabiano M. , Mei M. L. , Danovaro R.
Published: December 2019

Spatial and temporal changes in sedimentary nucleic acid concentrations in an abyssal locality of the northeastern Atlantic Ocean were investigated in relation to fluxes of nucleic acids produced in the photic layer. Sediment trap material, collected between 1996 and 1998 at depths of 1,000, 3,000, and 4,700 m, and sediment samples were analyzed for DNA and RNA content. Nucleic acid concentrations in the sediments were very high and displayed significant temporal changes, whereas mesoscale variability was low. DNA and RNA concentrations generally displayed opposite temporal patterns, which are likely to be dependent on the nature and characteristics of DNA and RNA molecules. Nucleic acid fluxes were high and displayed clear seasonal changes apparently coupled with seasonal pulses of primary production. However, while median values of DNA fluxes were relatively similar in all sediment traps, median values of RNA fluxes almost doubled from the 1,000- to the 4,700-m depth, suggesting differences in the metabolic activity of microbes associated with sinking particles. Significant relationships between DNA concentrations in the sediments and DNA fluxes and between RNA concentrations and RNA fluxes, indicating the presence of a clear pelagic-benthic coupling of particulate nucleic acids, were observed. The benthic system investigated was not steady state since we estimated that, from September 1996 to October 1998, nucleic acid concentration in the sediments decreased by about 165 mg of DNA m
−2
. Vertical profiles revealed a significant decrease in DNA concentration with depth in the sediments, reaching an asymptotic value of about 5 μg g
−1
. This DNA fraction constitutes a pool of potentially refractory DNA (accounting for 16 to 40% of the total DNA pool) that might be buried in the sediments.
Effects of Combined Shear and Thermal Forces on Destruction of
Microbacterium lacticum
Authors: Bulut S. , Waites W. M. , Mitchell J. R.
Published: December 2019

A twin-screw extruder and a rotational rheometer were used to generate shear forces in concentrated gelatin inoculated with a heat-resistant isolate of a vegetative bacterial species,
Microbacterium lacticum
. Shear forces in the extruder were mainly controlled by varying the water feed rate. The water content of the extrudates changed between 19 and 45% (wet weight basis). Higher shear forces generated at low water contents and the calculated die wall shear stress correlated strongly with bacterial destruction. No surviving microorganisms could be detected at the highest wall shear stress of 409 kPa, giving log reduction of 5.3 (minimum detection level, 2 × 10
4
CFU/sample). The mean residence time of the microorganism in the extruder was 49 to 58 s, and the maximum temperature measured in the end of the die was 73°C. The
D
75°C
of the microorganism in gelatin at 65% water content was 20 min. It is concluded that the physical forces generated in the reverse screw element and the extruder die rather than heat played a major part in cell destruction. In a rotational rheometer, after shearing of a mix of microorganisms with gelatin at 65% (wt/wt) moisture content for 4 min at a shear stress of 2.8 kPa and a temperature of 75°C, the number of surviving microorganisms in the sheared sample was 5.2 × 10
6
CFU/g of sample compared with 1.4 × 10
8
CFU/g of sample in the nonsheared control. The relative effectiveness of physical forces in the killing of bacteria and destruction of starch granules is discussed.
Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria
Authors: Gasol Josep M. , Zweifel Ulla Li , Peters Francesc , Fuhrman Jed A. , Hagström Åke
Published: December 2019

Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead
Bac
Light bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.
Hydrogen Profiles and Localization of Methanogenic Activities in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (
Cubitermes
spp.)
Authors: Schmitt-Wagner Dirk , Brune Andreas
Published: December 2019

It has been shown that the coexistence of methanogenesis and reductive acetogenesis in the hindgut of the wood-feeding termite
Reticulitermes flavipes
is based largely on the radial distribution of the respective microbial populations and relatively high hydrogen partial pressures in the gut lumen. Using Clark-type microelectrodes, we showed that the situation in
Cubitermes orthognathus
and other soil-feeding members of the subfamily Termitinae is different and much more complex. All major compartments of agarose-embedded hindguts were anoxic at the gut center, and high H
2
partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H
2
. Posterior to the P3 segment, however, H
2
concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H
2
was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H
2
or formate was added. Addition of H
2
to the gas headspace stimulated CH
4
emission of living termites, indicating that endogenous H
2
production limits methanogenesis also in vivo. At the low H
2
partial pressures in the posterior hindgut, methanogens would most likely outcompete homoacetogens for this electron donor. This might explain the apparent predominance of methanogenesis over reductive acetogenesis in the hindgut of soil-feeding termites, although the presence of homoacetogens in the anterior, highly alkaline region cannot yet be excluded. In addition, the direct contact of anterior and posterior hindgut compartments in situ permits a cross-epithelial transfer of H
2
or formate, which would not only fuel methanogenesis in these compartments, but would also create favorable microniches for reductive acetogenesis. In situ rates and spatial distribution of H
2
-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497–4505, 1999).
Diversity and Abundance of Bolidophyceae (Heterokonta) in Two Oceanic Regions
Authors: Guillou Laure , Moon-Van Der Staay Seung-Yeo , Claustre Herve , Partensky Frederic , Vaulot Daniel
Published: December 2019

The diversity and abundance of the Bolidophyceae (Heterokonta), a newly described picoplanktonic algal class which is a sister group to the diatoms, was assessed in the equatorial Pacific Ocean and in the Mediterranean Sea by culture isolation, molecular biology techniques, and pigment analyses. Eight strains of Bolidophyceae were isolated in culture from different mesotrophic and oligotrophic areas. The corresponding small subunit (SSU) rRNA gene sequences allowed us to design two probes specific for the Bolidophyceae. These probes have been used in natural samples (i) to selectively amplify and detect Bolidophyceae sequences and (ii) to quantify the relative abundance of Bolidophyceae within the picoeukaryote community. Sequences available to date indicate that the class Bolidophyceae comprises at least three different clades, two corresponding to the previously described species
Bolidomonas pacifica
and
Bolidomonas mediterranea
and the third one corresponding to a subspecies of
B. pacifica
. Amplification of the SSU rRNA gene from natural samples with universal primers and hybridization using a
Bolidomonas
-specific probe followed by a eukaryote-specific probe allowed us to estimate the contribution of the Bolidophyceae to the eukaryotic DNA in both Pacific and Mediterranean waters to be lower than 1%. Similarly, high-performance liquid chromatography analyses of fucoxanthin, the major carotenoid present in Bolidophyceae, indicated that less than 4% of the total chlorophyll
a
in the picoplanktonic fraction in the equatorial Pacific was due to Bolidophyceae. Consequently, although strains of Bolidophyceae have been isolated from samples collected at several stations, this new class seems to have been a minor component of the natural picoeukaryotic populations in the ecosystems investigated, at least during the periods sampled.
Mechanisms of Action of Carvacrol on the Food-Borne Pathogen
Bacillus cereus
Authors: Ultee A. , Kets E. P. W. , Smid E. J.
Published: December 2019

Carvacrol, a naturally occurring compound mainly present in the essential oil fraction of oregano and thyme, was studied for its effect on bioenergetic parameters of vegetative cells of the food-borne pathogen
Bacillus cereus
. Incubation for 30 min in the presence of 1 to 3 mM carvacrol reduced the viable cell numbers exponentially. Carvacrol (2 mM) significantly depleted the intracellular ATP pool to values close to 0 within 7 min. No proportional increase of the extracellular ATP pool was observed. Depletion of the internal ATP pool was associated with a change of the membrane potential (Δψ). At concentrations of 0.01 mM carvacrol and above, a significant reduction of Δψ was observed, leading to full dissipation of Δψ at concentrations of 0.15 mM and higher. Finally, an increase of the permeability of the cytoplasmic membrane for protons and potassium ions was observed (at 0.25 and 1 mM carvacrol, respectively). From this study, it could be concluded that carvacrol interacts with the membranes of
B. cereus
by changing its permeability for cations like H
+
and K
+
. The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.
Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses
Authors: Santegoeds Cecilia M. , Damgaard Lars Riis , Hesselink Gijs , Zopfi Jakob , Lens Piet , Muyzer Gerard , de Beer Dirk
Published: December 2019

Using molecular techniques and microsensors for H
2
S and CH
4
, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S
2−
m
−3
s
−1
or 2 × 10
−9
mmol s
−1
per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S
2−
m
−3
s
−1
or 7 × 10
−9
mol s
−1
per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH
4
m
−3
s
−1
or 10
−9
mmol s
−1
per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH
4
m
−3
s
−1
or 5 × 10
−9
mmol s
−1
per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H
2
), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and
Eubacteria
spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.
Biodegradation of Methyl
tert
-Butyl Ether by a Bacterial Pure Culture
Authors: Hanson Jessica R. , Ackerman Corinne E. , Scow Kate M.
Published: December 2019

A bacterial strain, PM1, which is able to utilize methyl
tert
-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 × 10
6
cells ml
−1
were 0.07, 1.17, and 3.56 μg ml
−1
h
−1
for initial concentrations of 5, 50, and 500 μg MTBE ml
−1
, respectively. When incubated with 20 μg of uniformly labeled [
14
C]MTBE ml
−1
, strain PM1 converted 46% to
14
CO
2
and 19% to
14
C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE
−1
. Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 μg of MTBE ml
−1
added to the core material. The rate of MTBE removal increased with additional inputs of 20 μg of MTBE ml
−1
. These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.
Relationships of Bradyrhizobia from the Legumes
Apios americana
and
Desmodium glutinosum
Authors: Parker Matthew A.
Published: December 2019

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with
Apios americana
,
Desmodium glutinosum
, and
Amphicarpaea bracteata
, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35
A. americana
and 33
D. glutinosum
isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in
A. bracteata
populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from
A. americana
and
D. glutinosum
were either identical to
A. bracteata
isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of
A. bracteata
plants inoculated with isolates from
D. glutinosum
were equivalent to levels found with native
A. bracteata
bacterial isolates, but none of the three
A. americana
isolates tested had high symbiotic effectiveness on
A. bracteata
. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both
A. americana
and
D. glutinosum
harbored rare bacterial genotypes similar to
Bradyrhizobium japonicum
USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to
Bradyrhizobium elkanii
.
Phylogenetic Diversity of Nitrogen Fixation Genes in the Symbiotic Microbial Community in the Gut of Diverse Termites
Authors: Ohkuma Moriya , Noda Satoko , Kudo Toshiaki
Published: December 2019

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (
nifH
) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated
nifH
genes revealed the presence of diverse
nifH
sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the
nifH
sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic
nif
group (consisting of clostridia and sulfur reducers) or the alternative
nif
methanogen group among the
nifH
phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent
nif
group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The
nifH
groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.
Regulation of Exopolysaccharide Production by
Lactococcus lactis
subsp.
cremoris
by the Sugar Source
Authors: Looijesteijn Petronella J. , Boels Ingeborg C. , Kleerebezem Michiel , Hugenholtz Jeroen
Published: December 2019

Lactococcus lactis
produced more exopolysaccharide (EPS) on glucose than on fructose as the sugar substrate, although the transcription level of the
eps
gene cluster was independent of the sugar source. A major difference between cells grown on the two substrates was the capacity to produce sugar nucleotides, the EPS precursors. However, the activities of the enzymes required for the synthesis of nucleotide sugars were not changed upon growth on different sugars. The activity of fructosebisphosphatase (FBPase) was by far the lowest of the enzymes involved in precursor formation under all conditions. FBPase catalyzes the conversion of fructose-1,6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose. By overexpression of the
fbp
gene, which resulted in increased EPS synthesis on fructose, it was proven that the low activity of FBPase is indeed limiting not only for EPS production but also for growth on fructose as a sugar source.
Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1
Authors: Schaefer Jeffra K. , Oremland Ronald S.
Published: December 2019

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 μM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [
14
C]formaldehyde to
14
CO
2
but had only a small capacity for oxidation of [
14
C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [
14
C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent

K
s

values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO
2
. The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.
Reactor-Scale Cultivation of the Hyperthermophilic Methanarchaeon
Methanococcus jannaschii
to High Cell Densities
Authors: Mukhopadhyay Biswarup , Johnson Eric F. , Wolfe Ralph S.
Published: December 2019

For the hyperthermophilic and barophilic methanarchaeon
Methanococcus jannaschii
, we have developed a medium and protocols for reactor-scale cultivation that improved the final cell yield per liter from ∼0.5 to ∼7.5 g of packed wet cells (∼1.8 g dry cell mass) under autotrophic growth conditions and to ∼8.5 g of packed wet cells (∼2 g dry cell mass) with yeast extract (2 g liter
−1
) and tryptone (2 g liter
−1
) as medium supplements. For growth in a sealed bottle it was necessary to add Se to the medium, and a level of 2 μM for added Se gave the highest final cell yield. In a reactor
M. jannaschii
grew without added Se in the medium; it is plausible that the cells received Se as a contaminant from the reactor vessel and the H
2
S supply. But, for the optimal performance of a reactor culture, an addition of Se to a final concentration of 50 to 100 μM was needed. Also, cell growth in a reactor culture was inhibited at much higher Se concentrations. These observations and the data from previous work with methanogen cell extracts (B. C. McBride and R. S. Wolfe, Biochemistry 10:4312–4317, 1971) suggested that from a continuously sparged reactor culture Se was lost in the exhaust gas as volatile selenides, and this loss raised the apparent required level of and tolerance for Se. In spite of having a proteinaceous cell wall,
M. jannaschii
withstood an impeller tip speed of 235.5 cms
−1
, which was optimal for achieving high cell density and also was the higher limit for the tolerated shear rate. The organism secreted one or more acidic compounds, which lowered pH in cultures without pH control; this secretion continued even after cessation of growth.
Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments
Authors: Costello Andria M. , Lidstrom Mary E.
Published: December 2019

The 16S rRNA and
pmoA
genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes
Msp
I,
Hae
III, and
Hha
I. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera
Methylomonas
,
Methylosinus
, and
Methylocystis
were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs,
pmoA
. Phylogenetic analyses of both the 16S rRNA and
pmoA
gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.
Natural Communities of
Achromatium oxaliferum
Comprise Genetically, Morphologically, and Ecologically Distinct Subpopulations
Authors: Gray N. D. , Howarth R. , Rowan A. , Pickup R. W. , Jones J. Gwyn , Head I. M.
Published: December 2019

The diversity and ecology of natural communities of the uncultivated bacterium
Achromatium oxaliferum
were studied by use of culture-independent approaches. 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as
A. oxaliferum
present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles). Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the
A. oxaliferum
communities present in all the sediments examined. Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified
A. oxaliferum
cells corresponded to genetically distinct
Achromatium
subpopulations. Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct. Furthermore, there was a high degree of endemism in the
Achromatium
sequences recovered from different sediments; identical sequences were never recovered from different sampling locations. In addition to ecological differences that were apparent between
Achromatium
communities from different freshwater sediments, the distribution of different subpopulations of
Achromatium
in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions. This result suggests that
Achromatium
populations have undergone adaptive radiation and that the divergent
Achromatium
species occupy different niches in the sediments which they inhabit.
Analyses of Spatial Distributions of Sulfate-Reducing Bacteria and Their Activity in Aerobic Wastewater Biofilms
Authors: Okabe Satoshi , Itoh Tsukasa , Satoh Hisashi , Watanabe Yoshimasa
Published: December 2019

The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O
2
, H
2
S, NO
2
−
, NO
3
−
, NH
4
+
, and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 10
9
to 10
10
cells per cm
3
of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained
Desulfobulbus
spp. were found to be numerically important members of SRB populations (approximately 10
8
to 10
9
cells per cm
3
). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 μm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S
0
) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 μm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate.
Reductive Dehalogenation and Conversion of 2-Chlorophenol to 3-Chlorobenzoate in a Methanogenic Sediment Community: Implications for Predicting the Environmental Fate of Chlorinated Pollutants
Authors: Becker Jennifer G. , Stahl David A. , Rittmann Bruce E.
Published: December 2019

Biotransformation of 2-chlorophenol by a methanogenic sediment community resulted in the transient accumulation of phenol and benzoate. 3-Chlorobenzoate was a more persistent product of 2-chlorophenol metabolism. The anaerobic biotransformation of phenol to benzoate presumably occurred via
para
-carboxylation and dehydroxylation reactions, which may also explain the observed conversion of 2-chlorophenol to 3-chlorobenzoate.
Epitope Identification for a Panel of Anti-
Sinorhizobium meliloti
Monoclonal Antibodies and Application to the Analysis of K Antigens and Lipopolysaccharides from Bacteroids
Authors: Reuhs Bradley L. , Stephens Samuel B. , Geller Daniel P. , Kim John S. , Glenn Joshua , Przytycki Jessica , Ojanen-Reuhs Tuula
Published: December 2019

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of
Sinorhizobium meliloti
were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506–509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654–661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-
S. meliloti
MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from
S. meliloti
and
Sinorhizobium fredii
. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from
S. meliloti
bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of
S. meliloti
NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the
S. meliloti
NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.
Ubiquity and Diversity of Dissimilatory (Per)chlorate-Reducing Bacteria
Authors: Coates John D. , Michaelidou Urania , Bruce Royce A. , O’Connor Susan M. , Crespi Jill N. , Achenbach Laurie A.
Published: December 2019

Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 × 10
3
to 2.4 × 10
6
cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type
c
cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 μmol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the
Proteobacteria
. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera
Pseudomonas
and
Azospirllum
. However, many were not closely related to any previously described organism and represented new genera within the
Proteobacteria
. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.
Characterization of Unexpected Growth of
Escherichia coli
O157:H7 by Modeling
Authors: Cornu Marie , Delignette-Muller Marie Laure , Flandrois Jean-Pierre
Published: December 2019

Modeling of batch kinetics in minimal synthetic medium was used to characterize
Escherichia coli
O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for
E. coli
K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other
E. coli
strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures,
E. coli
K-12 had a positive effect on
E. coli
O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for
E. coli
K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of
E. coli
K-12 on
E. coli
O157:H7 appeared to be similar to the effect of methionine.
Influence of Different Functional Elements of Plasmid pGT232 on Maintenance of Recombinant Plasmids in
Lactobacillus reuteri
Populations In Vitro and In Vivo
Authors: Heng Nicholas C. K. , Bateup Judith M. , Loach Diane M. , Wu Xiyang , Jenkinson Howard F. , Morrison Mark , Tannock Gerald W.
Published: December 2019

Plasmid pGT232 (5.1 kb), an indigenous plasmid of
Lactobacillus reuteri
100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein,
repA
. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an
L. reuteri
population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the
L. reuteri
population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.
Fate of pGFP-Bearing
Escherichia coli
O157:H7 in Ground Beef at 2 and 10°C and Effects of Lactate, Diacetate, and Citrate
Authors: Ajjarapu Srilatha , Shelef Leora A.
Published: December 2019

Although beef has been implicated in the largest outbreaks of
Escherichia coli
O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing
E. coli
O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10°C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2°C, the initial numbers of
E. coli
O157:H7 (3 and 5 log
10
CFU/g) decreased by ∼1 log
10
CFU/g when spoilage was manifest (>7 log
10
CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10°C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.