bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Gene Family Encoding Lipases in
Candida rugosa
by Competitive Reverse Transcription-PCR
Authors: Lee Guan-Chiun , Tang Shye-Jye , Sun Kuang-Hui , Shaw Jei-Fu 
Published: December 2019

ABSTRACT

Synthesis of multiple extracellular lipases in
Candida rugosa
has been demonstrated. However, it is difficult to characterize the expression spectrum of
lip
genes, since the sequences of the
lip
multigene family are very closely related. A competitive reverse transcription-PCR assay was developed to quantify the expression of
lip
genes. In agreement with the protein profile, the abundance of
lip
mRNAs was found to be (in decreasing order)
lip1
,
lip3
,
lip2
,
lip5
, and
lip4
. To analyze the effects of different culture conditions, the transcript concentrations for these mRNA species were normalized relative to the values for
gpd
, encoding glyceraldehyde-3-phosphate dehydrogenase. In relative terms,
lip1
and
lip3
were highly and constitutively expressed (about 10
5
molecules per μg of total RNA) whereas the other inducible
lip
genes, especially
lip4
, showed significant changes in mRNA expression under different culture conditions. These results indicate that differential transcriptional control of
lip
genes results in multiple forms of lipase proteins.
Construction of Environmental DNA Libraries in
Escherichia coli
and Screening for the Presence of Genes Conferring Utilization of 4-Hydroxybutyrate
Authors: Henne Anke , Daniel Rolf , Schmitz Ruth A. , Gottschalk Gerhard 
Published: December 2019

ABSTRACT

Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant
Escherichia coli
strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive
E. coli
clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These
E. coli
clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the
gbd
gene of
Ralstonia eutropha
, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (
orf1
and
orf6
) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced
orf1
gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced
orf6
gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.
Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of
Lactobacillus rhamnosus
and
L. casei
Strains
Authors: Tynkkynen Soile , Satokari Reetta , Saarela Maria , Mattila-Sandholm Tiina , Saxelin Maija 
Published: December 2019

ABSTRACT

A total of 24 strains, biochemically identified as members of the
Lactobacillus casei
group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for
L. rhamnosus
and
L. casei
identified the type strain
L. rhamnosus
ATCC 7469 and the neotype strain
L. casei
ATCC 334, respectively, but did not give any signal with the recently revived species
L. zeae
, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as
L. casei
. Our results are in accordance with the suggested new classification of the
L. casei
group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.
Glucose Metabolism and Kinetics of Phosphorus Removal by the Fermentative Bacterium
Microlunatus phosphovorus
Authors: Santos Margarida M. , Lemos Paulo C. , Reis Maria A. M. , Santos Helena 
Published: December 2019

ABSTRACT

Phosphorus and carbon metabolism in
Microlunatus phosphovorus
was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with
31
P and
13
C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment.
M. phosphovorus
fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, 3.34 mmol g of cell
−1
h
−1
and 1.56 mmol g of cell
−1
h
−1
, respectively, values only comparable with those determined in activated sludge. In the aerobic period, growth was strictly dependent on the availability of external phosphate. Natural abundance
13
C NMR showed the presence of reserves of glutamate and trehalose in cell suspensions. Unexpectedly, [1-
13
C]glucose was not significantly channeled to the synthesis of internal reserves in the anaerobic phase, and acetate was not during the aerobic stage, although the glutamate pool became labeled via the exchange with intermediates of the tricarboxylic acid cycle at the level of glutamate dehydrogenase. The intracellular pool of glutamate increased under anaerobic conditions and decreased during the aerobic period. The contribution of
M. phosphovorus
for phosphorus removal in wastewater treatment plants is discussed on the basis of the metabolic features disclosed by this study.
Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium
Methylocystis
sp. Strain WI 14
Authors: Grosse Stephan , Laramee Louise , Wendlandt Karin-Dagmar , McDonald Ian R. , Miguez Carlos B. , Kleber Hans-Peter 
Published: December 2019

ABSTRACT

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus
Methylocystis
. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α
2
β
2
γ
2
structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg
2+
ions (with a total loss of enzyme activity at 0.01 mM Hg
2+
) and Cu
2+
, Zn
2+
, and Ni
2+
ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from
Methylosinus trichosporium
OB3b,
Methylocystis
sp. strain M, and
Methylococcus capsulatus
(Bath).
Nested Allele-Specific PCR Primers Distinguish Genetic Groups of
Uncinula necator
Authors: Délye Christophe , Ronchi Valérie , Laigret Frédéric , Corio-Costet Marie-France 
Published: December 2019

ABSTRACT

Isolates of the obligately biotrophic fungus
Uncinula necator
cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of
U. necator
. We used the nucleotide sequences of the gene that encodes eburicol 14α-demethylase (
CYP51
) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations (three in
CYP51
and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the
CYP51
point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.
Phylogenetic Affiliation and Quantification of Psychrophilic Sulfate-Reducing Isolates in Marine Arctic Sediments
Authors: Sahm Kerstin , Knoblauch Christian , Amann Rudolf 
Published: December 2019

ABSTRACT

Thirteen psychrophilic sulfate-reducing isolates from two permanently cold fjords of the Arctic island Spitsbergen (Hornsund and Storfjord) were phylogenetically analyzed. They all belonged to the δ subclass of
Proteobacteria
and were widely distributed within this group, indicating that psychrophily is a polyphyletic property. A new 16S rRNA-directed oligonucleotide probe was designed against the largest coherent cluster of these isolates. The new probe, as well as a set of available probes, was applied in rRNA slot blot hybridization to investigate the composition of the sulfate-reducing bacterial community in the sediments. rRNA related to the new cluster of incompletely oxidizing, psychrophilic isolates made up 1.4 to 20.9% of eubacterial rRNA at Storfjord and 0.6 to 3.5% of eubacterial rRNA at Hornsund. This group was the second-most-abundant group of sulfate reducers at these sites. Denaturing gradient gel electrophoresis and hybridization analysis showed bands identical to those produced by our isolates. The data indicate that the psychrophilic isolates are quantitatively important in Svalbard sediments.
d
-Lactate Dehydrogenase Gene (
ldhD
) Inactivation and Resulting Metabolic Effects in the
Lactobacillus johnsonii
Strains La1 and N312
Authors: Lapierre Luciane , Germond Jacques-Edouard , Ott Andreas , Delley Michele , Mollet Beat 
Published: December 2019

ABSTRACT

Lactobacillus johnsonii
La1, a probiotic bacterium with demonstrated health effects, grows in milk, where it ferments lactose to
d
- and
l
-lactate in a 60:40% ratio. The
d
-lactate dehydrogenase (D-LDH) gene (
ldhD
) of this strain was isolated, and an in vitro-truncated copy of that gene was used to inactivate the genomic copy in two strains, La1 and N312, by gene replacement. For that, an 8-bp deletion was generated within the cloned
ldhD
gene to inactivate its function. The plasmid containing the altered
ldhD
was transferred to
L. johnsonii
via conjugative comobilization with
Lactococcus lactis
carrying pAMβ1. Crossover integrations of the plasmid at the genomic
ldhD
site were selected, and appropriate resolution of the cointegrate structures resulted in mutants that had lost the plasmid and in which the original
ldhD
was replaced by the truncated copy. These mutants completely lacked D-LDH activity. Nevertheless, the lower remaining L-LDH activity of the cells was sufficient to reroute most of the accumulating pyruvate to
l
-lactate. Only a marginal increase in production of the secondary end products acetaldehyde, diacetyl, and acetoin was observed. It can be concluded that in
L. johnsonii
D- and L-LDH are present in substantial excess for their role to eliminate pyruvate and regenerate NAD
+
and that accumulated pyruvate is therefore not easily redirected in high amounts to secondary metabolic routes.
Microbial Proline 4-Hydroxylase Screening and Gene Cloning
Authors: Shibasaki Takeshi , Mori Hideo , Chiba Shigeru , Ozaki Akio 
Published: December 2019

ABSTRACT

Microbial proline 4-hydroxylases, which hydroxylate free
l
-proline to
trans
-4-hydroxy-
l
-proline, were screened in order to establish an industrial system for biotransformation of
l
-proline to
trans
-4-hydroxy-
l
-proline. Enzyme activities were detected in eight strains, including strains of
Dactylosporangium
spp. and
Amycolatopsis
spp. The
Dactylosporangium
sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library of
Dactylosporangium
sp. strain RH1 constructed in
Escherichia coli
. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in
E. coli
as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The
E. coli
recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain,
Dactylosporangium
sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.
Identification of the
Pseudomonas stutzeri
OX1 Toluene–
o
-Xylene Monooxygenase Regulatory Gene (
touR
) and of Its Cognate Promoter
Authors: Arenghi Fabio L. G. , Pinti Marcello , Galli Enrica , Barbieri Paola 
Published: December 2019

ABSTRACT

Toluene–
o
-xylene monooxygenase is an enzymatic complex, encoded by the
touABCDEF
genes, responsible for the early stages of toluene and
o
-xylene degradation in
Pseudomonas stutzeri
OX1. In order to identify the loci involved in the transcriptional regulation of the
tou
gene cluster, deletion analysis and complementation studies were carried out with
Pseudomonas putida
PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and
o
-xylene and their corresponding phenols, of the toluene–
o
-xylene monooxygenase. A locus encoding a positive regulator, designated
touR
, was mapped downstream from the
tou
gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a
touA
-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (

P
ToMO

) was mapped, which showed the typical −24 TGGC, −12 TTGC sequences characteristic of ς
54
-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the
P. stutzeri
system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of
tou
structural genes in the absence of inducers. Basal toluene–
o
-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.
Radioactive Fingerprinting of Microorganisms That Oxidize Atmospheric Methane in Different Soils
Authors: Roslev Peter , Iversen Niels 
Published: December 2019

ABSTRACT

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with
14
CH
4
followed by analysis of radiolabelled phospholipid ester-linked fatty acids (
14
C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The
14
C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1ω8c (up to 9.0% of the total
14
C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1ω9, 18:1ω7, and 18:0). The
14
C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH
4
. The
14
C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the
14
C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.
Enhanced (+)-Catechin Transglucosylating Activity of
Streptococcus mutans
GS-5 Glucosyltransferase-D due to Fructose Removal
Authors: Meulenbeld Gerwin H. , Zuilhof Han , van Veldhuizen Adelbertus , van den Heuvel Robert H. H. , Hartmans Sybe 
Published: December 2019

ABSTRACT

The (+)-catechin transglucosylating activities of several glucosyltransferases (GTFs) from the genus
Streptococcus
were compared. For this purpose, a mixture of four GTFs from
Streptococcus sobrinus
SL-1 and recombinant GTF-B and GTF-D from
Streptococcus mutans
GS-5 expressed in
Escherichia coli
were studied. It was shown that after removal of α-glucosidase activity, GTF-D transglucosylated catechin with the highest efficiency. A maximal yield (expressed as the ratio of moles of glucoside formed to moles of catechin initially added) of 90% was observed with 10 mM catechin and 100 mM sucrose (

K
m

, 13 mM) in 125 mM potassium phosphate, pH 6.0, at 37°C.
1
H and
13
C nuclear magnetic resonance spectroscopy revealed the structures of two catechin glucosides, (+)-catechin-4′-
O
-α-
d
-glucopyranoside and (+)-catechin-4′,7-
O
-α-di-
d
-glucopyranoside. Fructose accumulation during glucosyl transfer from sucrose to the acceptor competitively inhibited catechin transglucosylation (

K
i

, 9.3 mM), whereas glucose did not inhibit catechin transglucosylation. The addition of yeasts was studied in order to minimize fructose inhibition by means of fructose removal. For this purpose, the yeasts
Pichia pastoris
and the mutant
Saccharomyces cerevisiae
T2-3D were selected because of their inabilities to utilize sucrose. Addition of
P. pastoris
or
S. cerevisiae
T2-3D to the standard reaction mixture resulted in a twofold increase in the duration of the maximum GTF-D transglucosylation rate. The addition of the yeasts also stimulated sucrose utilization by GTF-D.
Expression of the Isoamylase Gene of
Flavobacterium odoratum
KU in
Escherichia coli
and Identification of Essential Residues of the Enzyme by Site-Directed Mutagenesis
Authors: Abe Jun-ichi , Ushijima Chiaki , Hizukuri Susumu 
Published: December 2019

ABSTRACT

The isoamylase gene from
Flavobacterium odoratum
KU was cloned into and expressed in
Escherichia coli
JM109. The promoter of the gene was successful in
E. coli
, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same
M
r
, heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell. The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues. By changing five possible residues to Ala independently, it was found that Asp-374, Glu-422, and Asp-497 were essential. The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX. The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases. This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches.
On the Occurrence of Anoxic Microniches, Denitrification, and Sulfate Reduction in Aerated Activated Sludge
Authors: Schramm Andreas , Santegoeds Cecilia M. , Nielsen Helle K. , Ploug Helle , Wagner Michael , Pribyl Milan , Wanner Jiri , Amann Rudolf , de Beer Dirk 
Published: December 2019

ABSTRACT

A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge. Microsensor measurements (O
2
, NO
2
−
, NO
3
−
, and H
2
S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale. Incubations of activated sludge with
15
NO
3
−
and
35
SO
4
2−
were used to determine denitrification and sulfate reduction rates on a batch scale. In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low. However, in two sludges anoxia in flocs coincided with significant denitrification rates. Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions. In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase. A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels. This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges.