bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Fluxes in the Ammonium-Assimilatory Pathways in
Corynebacterium glutamicum
Studied by
15
N Nuclear Magnetic Resonance
Authors: Tesch M. , de Graaf A. A. , Sahm H.
Published: December 2019

Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in
Corynebacterium glutamicum
. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of
15
N label from [
15
N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo
15
N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different
C. glutamicum
strains in vivo. In both strains, transmembrane equilibration of 100 mM [
15
N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH
4
+
was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of
C. glutamicum
. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.
Anaerobic Degradation of Phthalate Isomers by Methanogenic Consortia
Authors: Kleerebezem Robbert , Pol Look W. Hulshoff , Lettinga Gatze
Published: December 2019

Three methanogenic enrichment cultures, grown on
ortho
-phthalate,
iso
-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (μ
S
max
) and biomass yields (Y

X
tot

S
) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for μ
S
max
and Y

X
tot

S
were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (K
S
) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate.
Stimulation of Strontium Accumulation in Linoleate-Enriched
Saccharomyces cerevisiae
Is a Result of Reduced Sr
2+
Efflux
Authors: Avery Simon V. , Smith Shareeka L. , Ghazi A. Mohamad , Hoptroff Michael J.
Published: December 2019

The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in
Saccharomyces cerevisiae
was investigated. Growth of
S. cerevisiae
in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to ∼70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr
2+
intracellularly at approximately twice the rate of
S. cerevisiae
grown without a fatty acid supplement. This effect was evident over a wide range of external Sr
2+
concentrations (25 μM to 5 mM) and increased with the extent of cellular 18:2 incorporation. Stimulation of Sr
2+
accumulation was not evident following enrichment of
S. cerevisiae
with either palmitoleate (16:1), linolenate (18:3) (
n
-3 and
n
-6 isomers), or eicosadienoate (20:2) (
n
-6 and
n
-9 isomers). Competition experiments revealed that Ca
2+
- and Mg
2+
-induced inhibition of Sr
2+
accumulation did not differ between unsupplemented and 18:2-supplemented cells. Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca
2+
-ATPase inhibitor), at a low concentration that precluded nonspecific K
+
efflux, increased intracellular Sr
2+
accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively. Thus, TFP abolished the enhanced Sr
2+
accumulation ability of 18:2-supplemented cells. Moreover, the rate of Sr
2+
release from Sr
2+
-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr
2+
-loaded 18:2-enriched cells. The influence of enrichment with other fatty acids on Sr
2+
efflux was variable. The results reveal an enhanced Sr
2+
accumulation ability of
S. cerevisiae
following 18:2-enrichment, which is attributed to diminished Sr
2+
efflux activity in these cells.
Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from
Trametes versicolor
Authors: Collins Patrick J. , O’Brien Margaret M. , Dobson Alan D. W.
Published: December 2019

The white rot basidiomycete
Trametes versicolor
secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from
T. versicolor
CU1, the transcript levels of which are repressed by low concentrations of Mn
2+
and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression.
Role of the
Trichoderma harzianum
Endochitinase Gene,
ech42
, in Mycoparasitism
Authors: Carsolio Carolina , Benhamou Nicole , Haran Shoshan , Cortés Carlos , Gutiérrez Ana , Chet Ilan , Herrera-Estrella Alfredo
Published: December 2019

The role of the
Trichoderma harzianum
endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42,
ech42
. We constructed several transgenic
T. harzianum
strains carrying multiple copies of
ech42
and the corresponding gene disruptants. The level of extracellular endochitinase activity when
T. harzianum
was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of
Rhizoctonia solani
cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against
R. solani
or
Sclerotium rolfsii
were observed in greenhouse experiments.
Antisense RNA Strategies for Metabolic Engineering of
Clostridium acetobutylicum
Authors: Desai Ruchir P. , Papoutsakis Eleftherios T.
Published: December 2019

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of
Clostridium acetobutylicum
. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by ∼100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.
Initial Reactions in the Biodegradation of 1-Chloro-4-Nitrobenzene by a Newly Isolated Bacterium, Strain LW1
Authors: Katsivela Eleftheria , Wray Victor , Pieper Dietmar H. , Wittich Rolf-Michael
Published: December 2019

Bacterial strain LW1, which belongs to the family
Comamonadaceae
, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by
1
H and
13
C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by
1
H and
13
C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.
Suppression of the Biocontrol Agent
Trichoderma harzianum
by Mycelium of the Arbuscular Mycorrhizal Fungus
Glomus intraradices
in Root-Free Soil
Authors: Green Helge , Larsen John , Olsson Pål Axel , Jensen Dan Funck , Jakobsen Iver
Published: December 2019

Trichoderma harzianum
is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between
Glomus intraradices
and
T. harzianum
in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal
33
P transport and β-glucuronidase (GUS) activity were used to monitor activity of
G. intraradices
and a GUS-transformed strain of
T. harzianum
, respectively. As growth and metabolism of
T. harzianum
are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for
T. harzianum
, was investigated. The presence of
T. harzianum
in root-free soil reduced root colonization by
G. intraradices
. The external hyphal length density of
G. intraradices
was reduced by the presence of
T. harzianum
in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal
33
P transport by
G. intraradices
also was not affected by
T. harzianum
. This suggests that
T. harzianum
exploited the dead mycelium but not the living biomass of
G. intraradices
. The presence of external mycelium of
G. intraradices
suppressed
T. harzianum
population development and GUS activity. Stimulation of the hyphal biomass of
G. intraradices
by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of
G. intraradices
were not affected by the antagonistic fungus
T. harzianum
; in contrast,
T. harzianum
was adversely affected by
G. intraradices
.
Regional Differences in Production of Aflatoxin B
1
and Cyclopiazonic Acid by Soil Isolates of
Aspergillus flavus
along a Transect within the United States
Authors: Horn B. W. , Dorner J. W.
Published: December 2019

Soil isolates of
Aspergillus flavus
from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B
1
and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The
A. flavus
isolates were identified as members of either the L strain (
n
= 774), which produces sclerotia that are >400 μm in diameter, or the S strain (
n
= 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B
1
, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B
1
production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B
1
and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B
1
per ml. The percentages of isolates producing >10 μg of aflatoxin B
1
per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B
1
or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.
Expression of the
Corynebacterium glutamicum panD
Gene Encoding
l
-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in
Escherichia coli
Authors: Dusch Nicole , Pühler Alfred , Kalinowski Jörn
Published: December 2019

The
Corynebacterium glutamicum panD
gene was identified by functional complementation of an
Escherichia coli panD
mutant strain. Sequence analysis revealed that the coding region of
panD
comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A defined
C. glutamicum panD
mutant completely lacked
l
-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The
C. glutamicum panD
(

panD
C.g.

) as well as the
E. coli panD
(

panD
E.c.

) genes were cloned into a bifunctional expression plasmid to allow gene analysis in
C. glutamicum
as well as in
E. coli
. The enhanced expression of

panD
C.g.

in
C. glutamicum
resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the

panD
C.g.

gene product is proteolytically processed into two subunits. By increased expression of

panD
C.g.

in
C. glutamicum
, the activity of
l
-aspartate-α-decarboxylase was 288-fold increased, whereas the

panD
E.c.

gene resulted only in a 4-fold enhancement. The similar experiment performed in
E. coli
revealed that

panD
C.g.

achieved a 41-fold increase and that

panD
E.c.

achieved a 3-fold increase of enzyme activity. The effect of the

panD
C.g.

and

panD
E.c.

gene expression in
E. coli
was studied with a view to pantothenate accumulation. Only by expression of the

panD
C.g.

gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the

panD
E.c.

gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression of

panD
C.g.

in
E. coli
yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)
−1
h
−1
.
Use of Green Fluorescent Protein To Detect Expression of an Endopolygalacturonase Gene of
Colletotrichum lindemuthianum
during Bean Infection
Authors: Dumas Bernard , Centis Sylvie , Sarrazin Nathalie , Esquerré-Tugayé Marie-Thérèse
Published: December 2019

The 5′ noncoding region of
clpg2
, an endopolygalacturonase gene of the bean pathogen
Colletotrichum lindemuthianum
, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that
clpg2
was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.
Diversity ofBacteroides fragilisStrains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater
Authors: Puig Anna , Queralt Nuria , Jofre Juan , Araujo Rosa
Published: December 2019

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains ofBacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 101to approximately 103phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.
Genetic Analysis of Biodegradation of Tetralin by a
Sphingomonas
Strain
Authors: Hernáez María José , Reineke Walter , Santero Eduardo
Published: December 2019

A strain designated TFA which very efficiently utilizes tetralin has been isolated from the Rhine river. The strain has been identified as
Sphingomonas macrogoltabidus
, based on 16S rDNA sequence similarity. Genetic analysis of tetralin biodegradation has been performed by insertion mutagenesis and by physical analysis and analysis of complementation between the mutants. The genes involved in tetralin utilization are clustered in a region of 9 kb, comprising at least five genes grouped in two divergently transcribed operons.
Biological and Molecular Detection of Toxic Lipodepsipeptide-Producing
Pseudomonas syringae
Strains and PCR Identification in Plants
Authors: Bultreys Alain , Gheysen Isabelle
Published: December 2019

Toxin-based identification procedures are useful for differentiating
Pseudomonas syringae
pathovars. A biological test on peptone-glucose-NaCl agar in which the yeast
Rhodotorula pilimanae
was used proved to be more reliable for detecting lipodepsipeptide-producing strains of
P. syringae
than the more usual test on potato dextrose agar in which
Geotrichum candidum
is used. A PCR test performed with primers designed to amplify a 1,040-bp fragment in the coding sequence of the
syrD
gene, which was assumed to be involved in syringomycin and syringopeptin secretion, efficiently detected the gene in pathovars that produce the lipodepsipeptides. Comparable results were obtained in both tests performed with strains of the syringomycin-producing organisms
P. syringae
pv. syringae,
P. syringae
pv. atrofaciens, and
P. syringae
pv. aptata, but the PCR test failed with a syringotoxin-producing
Pseudomonas fuscovaginae
strain. The specificity of the test was verified by obtaining negative PCR test results for related pathovars or species that do not produce the toxic lipodepsipeptides.
P. syringae
pv. syringae was detected repeatedly in liquid medium inoculated with diseased vegetative tissue and assayed by the PCR test. Our procedure was also adapted to detect
P. syringae
pv. morsprunorum with a
cfl
gene-based PCR test.
Sensitive Detection of
Escherichia coli
O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry
Authors: Pyle Barry H. , Broadaway Susan C. , McFeters Gordon A.
Published: December 2019

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen
Escherichia coli
O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown
E. coli
O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and
r
2
= 0.95 (
n
= 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and
r
2
= 0.98 (
n
= 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of
E. coli
O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.
Identification and Disruption of BetL, a Secondary Glycine Betaine Transport System Linked to the Salt Tolerance of
Listeria monocytogenes
LO28
Authors: Sleator Roy D. , Gahan Cormac G. M. , Abee Tjakko , Hill Colin
Published: December 2019

The trimethylammonium compound glycine betaine (
N
,
N
,
N
-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon
Listeria monocytogenes
. We report the identification of
betL
, a gene encoding a glycine betaine uptake system in
L. monocytogenes
, isolated by functional complementation of the betaine uptake mutant
Escherichia coli
MKH13. The
betL
gene is preceded by a consensus ς
B
-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from
Bacillus subtilis
(57% identity) and BetP from
Corynebacterium glutamicum
(41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed

K
m

value of 7.9 μM for glycine betaine uptake after heterologous expression of
betL
in
E. coli
MKH13 is consistent with values obtained for
L. monocytogenes
in other studies. In addition, a
betL
knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in
L. monocytogenes
. This mutant is also unable to withstand concentrations of salt as high as can the BetL
+
parent, signifying the role of the transporter in
Listeria
osmotolerance.
Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil
Authors: Chin Kuk-Jeong , Lukow Thomas , Conrad Ralf
Published: December 2019

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH
4
production rate, a decrease in the steady-state H
2
partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (
n
= 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families
Methanosarcinaceae
(185 bp) and
Methanosaetaceae
(284 bp), the kingdom
Crenarchaeota
(185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom
Euryarchaeota
(392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (
n
= 30) retrieved from the community were dominated by
Crenarchaeota
(70%), “novel
Euryarchaeota
” (23%), and
Methanosarcinacaeae
(7%). Further incubation at 30°C (
n
= 30 clones) resulted in a relative increase in members of the
Methanosarcinaceae
(77%), whereas further incubation at 15°C (
n
= 30 clones) resulted in a much more diverse community consisting of 33%
Methanosarcinaceae
, 23%
Crenarchaeota
, 20%
Methanosaetaceae
, and 17% novel
Euryarchaeota
. The appearance of
Methanosaetaceae
at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.
A Morphological and Molecular Perspective of
Trichoderma viride
: Is It One or Two Species?
Authors: Lieckfeldt Elke , Samuels Gary J. , Nirenberg Helgard I. , Petrini Orlando
Published: December 2019

Trichoderma
(
Ascomycetes
,
Hypocreales
) strains that have warted conidia are traditionally identified as
T. viride
, the type species of
Trichoderma
. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether
T. viride
comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to “true”
T. viride
, the anamorph of
Hypocrea rufa
. Type II represents a new species,
T. asperellum
, which is, in terms of molecular characteristics, close to the neotype of
T. hamatum
.
Inhibition of Nitrifiers and Methanotrophs from an Agricultural Humisol by Allylsulfide and Its Implications for Environmental Studies
Authors: Neufeld Josh D. , Knowles Roger
Published: December 2019

Allylsulfide, an inhibitor of ammonia monooxygenase, was tested to determine its ability to inhibit nitrification and methane oxidation in pure cultures, in agricultural humisol enrichment cultures, and in humisol slurries. We confirmed that allylsulfide is a differential inhibitor of cultures of nitrifiers and methanotrophs at concentrations of 1 and 200 μM, respectively, which result in 50% inhibition. However, although a nitrifying enrichment culture added to sterilized humisol was inhibited 50% by 4 μM allylsulfide, 500 μM allylsulfide was necessary for 50% inhibition of the endogenous nitrifying activity in nonsterile humisol. We concluded that native nitrifiers were protected, possibly by being in colonial aggregates or sheltered microenvironments.
The Carboxy-Terminal Portion of the Aflatoxin Pathway Regulatory Protein AFLR of
Aspergillus parasiticus
Activates
GAL1
::
lacZ
Gene Expression in
Saccharomyces cerevisiae
Authors: Chang Perng-Kuang , Yu Jiujiang , Bhatnagar Deepak , Cleveland Thomas E.
Published: December 2019

AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in
Aspergillus parasiticus
and
Aspergillus flavus
, as well as the sterigmatocystin synthesis genes in
Aspergillus nidulans
. We show here by fusion of various
aflR
coding regions to the
GAL4
DNA-binding coding region that the AFLR carboxyl terminus contained a region that activated
GAL1
::
lacZ
gene expression in
Saccharomyces cerevisiae
and that the AFLR internal region was required for the activation activity. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366, and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities. Strikingly, the Asp436His mutation abolished the activation activity. Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and 40% decreases in the activation activities, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the AFLR carboxy-terminal region is involved in transcription activation and that total acidity in this region is not a major determinant of AFLR’s activation ability in
S. cerevisiae
.
Effects of Larval Mosquitoes (Aedes triseriatus) and Stemflow on Microbial Community Dynamics in Container Habitats
Authors: Kaufman Michael G. , Walker Edward D. , Smith Tracy W. , Merritt Richard W. , Klug Michael J.
Published: December 2019

The dynamics of the microbial food sources forAedes triseriatuslarvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted fromPseudomonaceaetoEnterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.
Molecular Analysis of
Pseudomonas aeruginosa
: Epidemiological Investigation of Mastitis Outbreaks in Irish Dairy Herds
Authors: Daly M. , Power E. , Björkroth J. , Sheehan P. , O’Connell A. , Colgan M. , Korkeala H. , Fanning S.
Published: December 2019

Pseudomonas aeruginosa
is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the
P. aeruginosa
strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with
Cla
I and
Pvu
II enzymes. Eight of the clinical isolates gave the same
Cla
I ribotype pattern as the mastitis-causing strains. However,
Pvu
II proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same
P. aeruginosa
strain was responsible for the mastitis outbreaks in all 11 herds.
Characterization of the
meta
-Cleavage Compound Hydrolase Gene Involved in Degradation of the Lignin-Related Biphenyl Structure by
Sphingomonas paucimobilis
SYK-6
Authors: Peng Xue , Masai Eiji , Katayama Yoshihiro , Fukuda Masao
Published: December 2019

Sphingomonas paucimobilis
SYK-6 has the ability to transform a lignin-related biphenyl compound, 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), to 5-carboxyvanillic acid (5CVA) via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). In the 4.9-kb
Hin
dIII fragment containing the OH-DDVA
meta
-cleavage dioxygenase gene (
ligZ
), we found a novel hydrolase gene (
ligY
) responsible for the conversion of the
meta
-cleavage compound of OH-DDVA to 5CVA. Incorporation of
18
O from H
2
18
O into 5CVA indicated there was a hydrolytic conversion of the OH-DDVA
meta
-cleavage compound to 5CVA. LigY exhibited hydrolase activity only toward the
meta
-cleavage compound of OH-DDVA, suggesting its restricted substrate specificity.
Changes of Fermentation Pathways of Fecal Microbial Communities Associated with a Drug Treatment That Increases Dietary Starch in the Human Colon
Authors: Wolin Meyer J. , Miller Terry L. , Yerry Susan , Zhang Yongchao , Bank Shelton , Weaver Gary A.
Published: December 2019

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-
13
C]glucose and
12
CO
2
or with unlabeled glucose and
13
CO
2
, the distribution of
13
C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO
2
. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO
2
reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.
An Outbreak of Nonflocculating Catabolic Populations Caused the Breakdown of a Phenol-Digesting Activated-Sludge Process
Authors: Watanabe Kazuya , Teramoto Maki , Harayama Shigeaki
Published: December 2019

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter
−1
day
−1
and then to 1.5 g liter
−1
day
−1
. After the loading rate was increased to 1.5 g liter
−1
day
−1
, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter
−1
day
−1
. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and
gyrB
fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the
gyrB
phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific
gyrB
sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter
−1
day
−1
. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of
gyrB
-targeted fine population analyses in microbial ecology.
Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?
Authors: Pembrey Richard S. , Marshall Kevin C. , Schneider René P.
Published: December 2019

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited
Psychrobacter
sp. strain SW8 (glycocalyx-bearing cells),
Escherichia coli
(gram-negative cells without a glycocalyx), and
Staphylococcus epidermidis
(gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 ×
g
) modified many cell surface parameters significantly, although cell viability was considerably affected only in
E. coli
. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.
Survival of Low-pH Stress by
Escherichia coli
O157:H7: Correlation between Alterations in the Cell Envelope and Increased Acid Tolerance
Authors: Jordan Kieran N. , Oxford Lynn , O’Byrne Conor P.
Published: December 2019

Survival of a nontoxigenic isolate of
Escherichia coli
O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 × 10
4
CFU ml
−1
were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.
Photosynthetic Bradyrhizobia from
Aeschynomene
spp. Are Specific to Stem-Nodulated Species and Form a Separate 16S Ribosomal DNA Restriction Fragment Length Polymorphism Group
Authors: Molouba Flore , Lorquin Jean , Willems Anne , Hoste Bart , Giraud Eric , Dreyfus Bernard , Gillis Monique , de Lajudie Philippe , Masson-Boivin Catherine
Published: December 2019

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated
Aeschynomene
species
A. elaphroxylon
,
A. uniflora
, or
A. schimperi
and 69 root or stem nodule isolates from the stem-nodulated
Aeschynomene
species
A. afraspera
,
A. ciliata
,
A. indica
,
A. nilotica
,
A. sensitiva
, and
A. tambacoundensis
from various places in Senegal. These isolates, together with 45 previous isolates from various
Aeschynomene
species, were studied for host-specific nodulation within the genus
Aeschynomene
, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732–734, 1985). The whole collection of
Aeschynomene
nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of
puf
genes in photosynthetic
Aeschynomene
isolates was evidenced both by Southern hybridization with a
Rhodobacter capsulatus
photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45
Aeschynomene
isolates, including strain BTAi1, and 19 reference strains from
Bradyrhizobium japonicum
,
Bradyrhizobium elkanii
, and other
Bradyrhizobium
sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic
Aeschynomene
nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the
Bradyrhizobium
rRNA lineage, distinct from
B. japonicum
and
B. elkanii
.